1,720,966 research outputs found
Analysis of the molecular mechanisms of photoprotection in higher plants and green algae
No full textGli organismi fotosintetici verdi, piante e alghe, possiedono sofisticati meccanismi preposti alla
difesa dei propri apparati fotosintetici dai danni ossidativi fotoindotti. Tali meccanismi protettivi
funzionano tramite l’azione di pigmenti peculiari, le xantofille, coordinati alle proteine
dell’apparato fotosintetico appartenenti alla grande famiglia Lhc.
In questo lavoro di tesi mi sono occupata tramite tecniche fisiologiche, biochimiche e genetiche
dello studio dell’importante meccanismo fotoprotettivo “Non-Photochemical quenching” (NPQ),
utilizzato dagli organismi verdi allo scopo di controllare la concentrazione delle clorofille eccitate
allo stato di singoletto e limitarne l’eccesso, tramite un meccanismo a “feedback” di dissipazione
dell’energia sotto forma di calore.
Gli organismi utilizzati in questo studio sono stati Arabidopsis thaliana, modello per lo studio delle
piante superiori, e Chlamydomonas reinhardtii, modello per lo studio delle microalghe verdi.
Si è evidenziato come, nonostante entrambi gli organismi possiedano e utilizzino questo
meccanismo fotoprotettivo, vi siano notevoli differenze sia a livello quantitativo nella capacità di
dissipare termicamente l’eccesso di energia sia a livello qualitativo nelle modalità di innesco e
regolazione della dissipazione stessa.
Nel capitolo numero 1 intitolato “Tra fotosintesi e fotoinibizione: il ruolo fondamentale di
carotenoidi e proteine che legano carotenoidi nella fotoprotezione” viene fornita una panoramica
generale dell’argomento fotoprotezione in piante superiori e alghe verdi. Lo scopo di questa parte
della tesi è illustrare quali sono gli eventi che portano alla formazione di specie reattive
dell’ossigeno e quali sono i principali meccanismi messi in atto dagli organismi fotosintetici allo
scopo di detossificare le specie reattive dell’ossigeno o prevenirne la formazione.
Il Non-Photochemical Quenching appartiene alla seconda classe di meccanismi fotoprotettivi e
viene qui descritto a livello generale, utilizzando le più importanti informazioni comparse in
letteratura negli ultimi anni e dettagliando tutte le componenti di questo complesso meccanismo.
Il capitolo numero 2 intitolato “Interazioni tra la subunità del fotosistema II PsbS e le xantofille
secondo studi in vivo e in vitro” è dedicato ad uno studio dettagliato del meccanismo molecolare di
“quenching” nelle piante superiori tramite lo studio in vitro della proteina ricombinante PsbS e la
caratterizzazione fisiologica in vivo di mutanti di A. thaliana difettivi nella capacità di NPQ.
Essendo la dissipazione termica dipendente dalla presenza/sintesi delle xantofille luteina e
zeaxantina e dalla subunità del fotosistema II PsbS, si è andati a verificare in questo lavoro l’ipotesi
che l’interazione diretta di tale subunità con le xantofille fosse responsabile del fenomeno. Lo
studio ha dimostrato come PsbS non interagisca direttamente con tali pigmenti bensì vada ad
attivare il meccanismo agendo come sensore dello stato di sovra-eccitazione del sistema e dando il
via, tramite l’induzione di cambiamenti conformazionale nelle altre subunità del sistema antenna, al
processo di dissipazione termica svolto queste altre proteine fisicamente coordinate ai pigmenti.
Il terzo capitolo, “Non-Photochemical Quenching della fluorescenza delle clorofille in
Chlamydomonas reinhardtii” è una caratterizzazione comparativa della capacità di NPQ nell’alga
verde unicellulare Chlamydomonas reinhardtii utilizzando come termine di paragone le piante
superiori, in cui il meccanismo è più studiato e meglio conosciuto. In questo lavoro si è verificato
come la capacità di NPQ sia minore in Chlamydomonas rispetto alle piante superiori e osservabile a
livelli rilevanti solo in particolari condizioni di sovra-eccitazione della catena di trasporto
elettronico fotosintetica. Questo a dimostrazione di come tale strategia sia diversamente impiegata
dai due organismi e diversamente regolata.
Nel quarto capitolo, “La presenza del prodotto genico PsbS in Chlamydomonas reinhardtii e altri
organismi fotosintetici e la sua correlazione con il quenching dell’energia”, viene effettuato uno
studio più dettagliato delle differenze osservate tra piante superiori e alghe verdi, elucidando come
il prodotto genico PsbS, responsabile dell’attivazione del meccanismo di dissipazione termica in
piante superiori, non sia espresso nell’alga unicellulare benchè sia presente una putativa sequenza
genica. La sequenza genica corrispondente quando sovra-espressa non sembra codificare un
polipeptide funzionale, confermando come il meccanismo di NPQ sia radicalmente diverso tra
alghe e piante. L’analisi della presenza del prodotto genico PsbS in un gruppo di organismi
rappresentanti la maggior parte delle classi del regno Viridiplantae, accoppiata alla misura della loro
capacità di dissipazione termica, ha evidenziato come il meccanismo di NPQ PsbS-dipendente i) sia
chiaramente presente come strategia fotoprotettiva d’elezione tra le piante terrestri o Embriofite; ii)
sia conservato escusivamente in quelle alghe verdi pluricellulari adattate ad ambienti con elevati
livelli di luce intensa e variabile, iii) sia del tutto assente nelle microalghe unicellulari, in grado di
adottare altri meccanismi fotoprotettivi, o meccanismi dissipativi anche di notevole rilevanza ma
diversamente regolati a livello molecolare.The green photosynthetic organisms, plants and algae, have complex mechanisms in order to defend
their photosynthetic apparatus from photoinduced oxydative damages. Such protective mechanisms
work through the action of peculiar pigments, the xanthophylls, coordinated with the proteins of the
photosynthetic system, which own to the Lhc super-family.
In this thesis work I focused through physiological, biochemical and genetic techniques on the
characterisation of the important photoprotective mechanism “Non-Photochemical quenching”
(NPQ), used by the green organisms in order to control singlet excited chloropylls concentration
and limit their excess through a “feedback” mechanism of thermal dissipation of the excess energy.
The organisms used in this study have been Arabidopsis thaliana, model for studying higher plants,
and Chlamydomonas reinhardtii, model for studying green microalgae.
It has been highlighted how, even if both organisms have and use this protective mechanism, many
differences are present both at quantitative level in thermal dissipation capacity and at qualitative
level in the triggering and in the regulation of the dissipation.
In chapter number 1 entitled “In between photosynthesis and photoinhibition: the fundamental role
of carotenoids and carotenoid-binding proteins in photoprotection” a general view of
photoprotection in plants and algae is given. The aim of this part of the thesis is to illustrate the
events that bring to the formation of reactive oxigen species and which are the main mechanisms
put in action by the photosynthetic organisms in order to detoxify reactive oxygen species or to
prevent their formation.
The Non-Photochemical Quenching owns to this second class of photoprotective mechanisms and is
here described at general level, using the most importants informations found in literature in the last
years and detailing each component of this complex mechanism.
Chapter number 2 entitled “Interactions between the photosystem II subunit Psbs and xanthophylls
studied in vivo and in vitro” is devoted to a detailed study of the molecular mechanism of
quenching in higher plants through the in vitro analysis of the recombinant PsbS protein and the in
vivo physiological characterisation of A. thaliana mutants defective in NPQ capacity. Being the
thermal dissipation dependent on the presence/synthesis of xanthophylls lutein and zeaxanthin and
on the photosystem II PsbS, I checked the hypothesis that the direct interaction of this subunit with
xanthophylls was responsible for the phenomenon. The work demonstrated how PsbS does not
interact directly with such pigments, but activates the mechanism working as a molecular sensor of
the over-excited state of the system, and triggering, through conformational changes in other
antenna subunits, the process of thermal dissipation which occurs on these other proteins and their
coordinated pigments.
The third chapter, “Non-Photochemical Quenching of chlorophyll fluorescence in Chlamydomonas
reinhardtii” is a comparative characterisation of the NPQ capacity in the unicellular green alga
Chlamydomonas reinhardtii using higher plants as term of comparison, in which the mechanism is
more studied and better understood. In this work we report how the NPQ capacity is lower in
Chlamydomonas than in higher plants and detectable at reasonable levels only in particular
conditions of over-excitation of the photosynthetic electron chain. This demonstrates how such
strategy is differentialy adopted by these two organisms and differentially regulated.
In the fourth chapter, “The occurrence of the PsbS gene product in Chlamydomonas reinhardtii and
other photosynthetic organisms and its correlation with energy quenching”, I carried on a more
detailed study of the differences observed between higher plants and green algae, elucidating how
the gene product PsbS, responsible fot the activation of the thermal dissipation mechanism in higher
plants, is not expressed in the unicellular alga Chlamydomonas although a putative gene sequence is
present. The corresponding gene sequence, when over-expressed, does not seem to code a
functional polypeptide, confirming how the NPQ mechanism is radically different between plants
and algae. The analysis of the presence of PsbS protein in a group of organisms representative for
most of classes belonging to the Viridiplantae kingdom, coupled with the measure of their thermal
dissipation, evidenced how the PsbS-dependent NPQ-mechanism: i) is clearly present as favourite
photoprotective strategy among land plants or Embriophytes; ii) is exclusively conserved in those
multicellular green algae adapted to environments with higher and more variable light intensities;
iii) is completely absent in the unicellular microalgae, which adopt other photoprotective strategies,
or other types of relevant dissipative mechanisms which are differentially regulated at molecular
level
IN BETWEEN PHOTOSYNTHESIS AND PHOTOINHIBITION: THE FUNDAMENTAL ROLE OF CAROTENOIDS AND CAROTENOID-BINDING PROTEINS IN PHOTOPROTECTION
No full textRetrograde signaling and photoprotection in a gun4 mutant of Chlamydomonas reinhardtii.
No full textGUN4 is a regulatory subunit of Mg-chelatase involved in the control of tetrapyrrole synthesis in plants and cyanobacteria. Here, we report the first characterization of a gun4 insertion mutant of the unicellular green alga Chlamydomonas reinhardtii. The mutant contains 50% of chlorophyll as compared to wild-type and accumulates ProtoIX. In contrast to the increase in LHC transcription, the accumulation of most LHC proteins is drastically diminished, implying posttranscriptional down-regulation in the absence of transcriptional coordination. We found that 803 genes change their expression level in gun4 as compared to wild-type, by RNA-Seq, and this wide-ranging effect on transcription is apparent under physiological conditions. Besides LHCs, we identified transcripts encoding enzymes of the tetrapyrrole pathway and factors involved in signal transduction, transcription, and chromatin remodeling. Moreover, we observe perturbations in electron transport with a strongly decreased PSI-to-PSII ratio. This is accompanied by an enhanced activity of the plastid terminal oxidase (PTOX) that could have a physiological role in decreasing photosystem II excitation pressure
Interactions between the photosystem II subunit PsbS and xanthophylls studied in vivo and in vitro
No full textThe photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type
LHCBM1 and LHCBM2/7 polypeptides, components of the major LHCII complex, have distinct functional roles in the photosynthetic antenna system of Chlamydomonas reinhardtii
No full textThe photosystem II antenna of Chlamydomonas reinhardtii is composed of monomeric and trimeric complexes, the latter encoded by LHCBM genes. We employed artificial microRNA technology to specifically silence the LHCBM2 and LHCBM7 genes, encoding identical mature polypeptides, and the LHCBM1 gene. As a control, we studied the npq5 mutant, deficient in the LHCBM1 protein. The organization of LHCII complexes, functional antenna size, capacity for photoprotection, thermal energy dissipation and state transitions, and resistance to reactive oxygen species was studied in the various genotypes. Silencing of the LHCBM2/7 genes resulted in a decrease of an LHCII protein with an apparent molecular mass of 22 kDa, whereas silencing/lack of LHCBM1 caused the decrease/disappearance of a 23-kDa protein. A decrease in the abundance of trimeric LHCII complexes and in functional antenna size was observed in both LHCBM2/7 and LHCBM1 knockouts. In agreement with previous data, depletion of LHCBM1 decreased the capacity for excess energy dissipation but not the ability to perform state transitions. The opposite was true for LHCBM2/7, implying that this polypeptide has a different functional role from LHCBM1. The abundance of LHCBM1 and LHCBM2/7 is in both cases correlated with resistance to superoxide anion, whereas only LHCBM1 is also involved in singlet oxygen scavenging. These results suggest that different LHCBM components have well defined, non-redundant functions despite their high homology, implying that engineering of LHCBM proteins can be an effective strategy for manipulating the light harvesting system of Chlamydomonas reinhardtii
Mutagenesis and phenotypic selection as a strategy toward domestication of Chlamydomonas reinhardtii strains for improved performance in photobioreactors
No full textAcclimation of Chlamydomonas reinhardtii to different growth irradiances
No full textWe report on the changes the photosynthetic apparatus of Chlamydomonas reinhardtii undergoes upon acclimation to different light intensity. When grown in high light, cells had a faster growth rate and higher biomass production compared with low and control light conditions. However, cells acclimated to low light intensity are indeed able to produce more biomass per photon available as compared with high light-acclimated cells, which dissipate as heat a large part of light absorbed, thus reducing their photosynthetic efficiency. This dissipative state is strictly dependent on the accumulation of LhcSR3, a protein related to light-harvesting complexes, responsible for nonphotochemical quenching in microalgae. Other changes induced in the composition of the photosynthetic apparatus upon high light acclimation consist of an increase of carotenoid content on a chlorophyll basis, particularly zeaxanthin, and a major down-regulation of light absorption capacity by decreasing the chlorophyll content per cell. Surprisingly, the antenna size of both photosystem I and II is not modulated by acclimation; rather, the regulation affects the PSI/PSII ratio. Major effects of the acclimation to low light consist of increased activity of state 1 and 2 transitions and increased contributions of cyclic electron flow
Nonphotochemical Quenching of Chlorophyll Fluorescence in Chlamydomonas reinhardtii
No full textUnlike plants, Chlamydomonas reinhardtii shows a restricted ability to develop nonphotochemical quenching upon illumination. Most of this limited quenching is due to state transitions instead of DeltapH-driven high-energy state quenching, qE. The latter could only be observed when the ability of the cells to perform photosynthesis was impaired, either by lowering temperature to approximately 0 degrees C or in mutants lacking RubisCO activity. Two main features were identified that account for the low level of qE in Chlamydomonas. On one hand, the electrochemical proton gradient generated upon illumination is apparently not sufficient to promote fluorescence quenching. On the other hand, the capacity to transduce the presence of a DeltapH into a quenching response is also intrinsically decreased in this alga, when compared to plants. The possible mechanism leading to these differences is discussed
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
- …
