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Regulation of light harvesting in plants: structural and functional studies of the Violaxanthin De-epoxidase
No full textGli organismi fotosintetici , come piante ed algae. hanno bisogno dell’energia luminosa per la loro
sopravvivenza tuttavia le condizioni ambientali sono estremamente variabili e questi devono riuscire a
rispondere a breve e a lungo termine ai diversi stimoli: quando la luce esterna è debole l’assorbimento
luminoso deve essere potenziato, mentre in condizioni di alta luminosità, l’energia in eccesso deve essere
dissipata, evitando così la formazione di specie radicaliche, dannose per le cellule.
I carotenoidi sono molecole con un ruolo chiave nella risposta alle diverse condizioni ambientali, essendo
capaci di dissipare le potenziali specie reattive. In aggiunta a ciò, quando l’illuminazione è in eccesso, si
attiva la sintesi della zeaxantina, un carotenoide particolarmente efficiente nella foto protezione. Quando
la luce ritorna nelle condizioni normali, zeaxantina è riconvertita a violaxantina. Quindi, in relazione alle
diverse condizioni di illuminazione, c’e’ una conversione tra le due specie, creando il cosi’ detto ciclo delle
xantofille, che è capace di modulare l’efficienza di assorbimento da parte dell’apparato fotosintetico.
Gli enzimi coinvolti nel ciclo delle xantofille sono la violaxantina de‐eposidasi (VDE) e la zeaxantina
eposidasi (ZE), che catalizzano rispettivamente la sintesi e la degradazione della zeaxantina. Lo scopo di
questo lavoro fu quello di capire il meccanismo molecolare della regolazione del ciclo delle xantofille, dallo
studio delle proprietà biochimiche e strutturali di questi due enzimi.
L’attenzione fu particolarmente rivolta all’enzima VDE, mentre solo un lavoro preliminare fu fatto sulla ZE
(appendice 1). Nel caso della VDE, invece, fummo in grado di ottenere i primi dati strutturali sull’enzima,
risolvendo la struttura terziaria del dominio lipocalinico della proteina che rappresenta metà della proteina.
Grazie a questi dati siamo riusciti a dimostrare come la proteina cambia conformazione in funzione del pH.
I dati strutturali raccolti danno inoltre un’idea di quale possa essere il sito di legame dell’enzima, anche
identificando i residui coinvolti nella reazione. Infine nel capitolo 3 analizziamo il ruolo delle rimanenti parti
dell’enzima nelle loro funzioni: il risultato più evidente consiste nell’aver identificato il dominio C‐terminale,
ricco in residui Gluttammici, fondamentale per l’associazione della proteina alle membrane tilacoidali.
In appendice 2, invece, è presentato il lavoro preliminare sul licopene β ciclasi, un enzima della biosintesi
dei carotenoidi. Abbiamo espresso e purificato la proteina ricombinante per produrre un anticorpo
specifico che riconoscesse l’enzima. Quest’ultimo lavoro è parte di un più ampio progetto, rivolto a
studiare la regolazione della biosintesi dei carotenoidi nelle piante in risposta alle condizioni ambientali.Plants are sessile organisms: when environmental conditions change, they cannot move but have to
respond by adapting their metabolism. This is particularly challenging with respect to illumination:
harvesting efficiency must be maximized when light is limiting while in high light conditions all
energy in excess has to be dissipated to avoid the fast formation of radical species harmful for the
cell.
Carotenoids are molecules with a key role in the response to environmental conditions, being able
to safely dissipate potentially reactive excited species. In addition to this general ability of all
carotenoids, when illumination is in excess there is the activation of the synthesis of zeaxanthin, a
carotenoid particularly efficient in photoprotection. When light is back to normal, zeaxanthin is
back converted into violaxanthin. Depending on illumination conditions, thus, there is a conversion
between these two species, forming the so called xanthophyll cycle, which are modulating the light
harvesting efficiency of photosynthetic apparatus.
Enzymes involved in the xanthophyll cycle are the violaxanthin de-epoxidase (VDE) and
zeaxanthin Epoxidase (ZE), which are catalyzing respectively the synthesis and degradation of
zeaxanthin. The aim of this work was to understand the molecular mechanisms of the regulation of
the xanthophyll cycle, by studying the biochemical and structural properties of these two enzymes.
The attention was particularly focused on VDE, while only preliminary work was done on ZE
(appendix 1). In the case of VDE, instead, we were able to obtain the first structural information on
the enzyme, resolving the 3D structure of the lipocalin like domain of the protein, representing half
of the full protein. Thanks to these data we were able to show how the protein in undergoing a pH
dependent conformational change. Structural data also provided suggestions on the violaxanthin
binding site to the enzyme. This hypothesis was tested in the chapter 2 by site directed mutagenesis.
Merging structural and mutational data we were able to propose a confident model of violaxanthin
binding to the enzyme, also identifying residues involved in the catalytic reaction. Finally in chapter
3 we analyzed the role of the remaining part of the protein for the protein function: major result was
the finding that the C terminal domain, rich in Glu residues, is important for the association of the
enzyme to the thylakoids membrane.
In appendix 2 is instead presented some preliminary work on lycopene β-cyclase, an enzyme of the
carotenoid biosynthetic pathway. We expressed and purified the recombinant protein to produce a
specific antibody recognizing the enzyme as part of a wider project aiming to study the regulation
of carotenoid biosynthesis in plants in response to environmental conditions
A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana
Get PDFPlants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin beta-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate
Mutation Analysis of the Violaxanthin de-epoxidase identifies substrate binding sites and residues involved in catalysis
No full textPlants are able to deal with variable environmental conditions: when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by Violaxanthin De-Epoxidase (VDE), using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the VDE catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analysed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate binding sites and point to two residues, D177 and Y198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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