Biochemistry of Rubisco……………

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Biochemistry of Rubisco…………… Departament de Biologia Facultat de Ciències Ecophysiological traits and their responses to drought in species from the Balearic Islands with different growth forms PhD Thesis Jeroni Galmés Galmés BSc. Biological Sciences (University of the Balearic Islands) Mallorca, 2006 Supervisor: Dr. Jaume Flexas Sans Jo, Dr. JAUME FLEXAS SANS, Professor Titular d’Escola Universitària de la Facultat de Ciències de la Universitat de les Illes Balears, CERTIFICO: Que Jeroni Galmés Galmés ha realitzat sota la meva direcció, en el Grup de Recerca en Biologia de les Plantes en Condicions Mediterrànies, de la Facultat de Ciències de la Universitat de les Illes Balears, el treball que, per optar al grau de Doctor en Ciències (Biologia), presenta amb el títol: “Ecophysiological traits and their responses to drought in species from the Balearic Islands with different growth forms” Considerant finalitzada la present memòria, autoritzo la seva presentació per a que pugui ésser jutjada pel tribunal corresponent. Per a que així consti, signo el present certificat a Palma, el 19 de gener de 2006. Jaume Flexas Sans L’EXTENSIÓ DE TERRA QUE CONTEMPLES PREN-LA COM UN REFLEX DE TU MATEIX, VULGUES CONFONDRE’T AMB ELS SEUS EXEMPLES, LA TERRA I TU BLEIXEU EN UN SOL BLEIX (RAMON LLULL) Acknowledgements AGRAÏMENTS – ACKNOWLEDGEMENTS Al llarg dels anys dedicats a la realització d’aquesta tesi han estat moltes les persones que de qualque manera m’han ajudat per a que aquest treball es pogués dur a terme. Amb tots ells estic en deute per la seva dedicació. En primer lloc a tots els membres del grup investigador en el qual he desenvolupat la meva tasca com a estudiant de doctorat, i que han col·laborat en la realització del treball experimental, en la redacció de les publicacions associades o simplement en crear un bon ambient de treball, gràcies a la seva agradable companyia. Sempre han estat allà quan se’ls ha demanat una mà. A na Josefina Bota li vull agrair la seva ajuda en la posada a punt d’algunes de les tècniques desenvolupades. A U Pep Cifre per ajudar-me en l’anàlisi estadística i per confiar en mi en molts altres aspectes. A en Miquel Ribas, per ajudar-me en les mil qüestions tècniques que m’anaven sorgint en el dia a dia. En Xavi Gulías me va introduir en l’ús del Li-Cor 6400 i me va transmetre tot el seu interès i sabre sobre plantes mediterrànies. A en Maurici Mus i en Miquel Àngel Conesa els agreisc el seu inestimable suport tècnic sobre temes botànics. A n’Hipólito Medrano, l’amo, perquè ara ja fa un temps me va introduir aquest cuquet de la investigació. També per confiar en mi, acollint-me al seu grup d’investigació i fent possible la realització d’aquest projecte i pels seus inestimables consells científics. També a la resta companys i companyes de laboratori i de can bum amb els que he coincidit: na Maite, en Diego, na Cecilia, na Magdalena, n’Alícia, en Pepe, na Belen, en Toni, na Conchi, en Jeroni, na Margalida, així com a la resta de professors i investigadors de l’àrea: na Cata i en John, en Jaume Vadell i en Toni Bennàssar. Gràcies pel vostre bon rollo i companyia diària, que sense cap dubte ha fet que tota la feina sigui molt més bona de dur. El meu agraïment més especial per un autèntic crack de la fisiologia vegetal, en Jaume Flexas, director d’aquesta tesi doctoral. La seva ja enorme experiència, coneixements i capacitat resolutiva per a tot, trivialitzant tots els problemes que anaven sorgint, ha resultat vital en tot moment per a la realització i anàlisi del treball dut a terme arrel de les investigacions. i Acknowledgements I would also like to acknowledge Martin Parry’s research group for allowing me to undertake part of this project at the Rothamsted Experimental Station, in Harpenden. Fiona Gilzean made possible the growth and survival of Mediterranean species in England. Pippa Madgwick and Richard Haslam supervised experiments on molecular biology. John Andraloj helped in technical questions in British labs. With my deepest admiration I would also like to share part of this work with someone very special to me, an extraordinary person and a great scientist, Alfred Keys. Although from a distance, Alf has been by my side, supporting and advising me when troubles in the lab became bigger. All you, together with unforgettable friends from the Manor, made funnier my stages in the cloudy and cold English winters, far away from the ‘Thistlefield’. Al llarg d’aquests anys també han estat moltes les persones que, a part de la UIB i de la Rothamsted Experimental Station, m’han ajudat en la meva formació com a investigador. El grup de na Monona Abadía i en Fermín Morales, de l’Aula Dei de Saragossa, el grup de n’Albert Mas, de la Universitat Rovira i Virgili de Tarragona, i el grup d’Andrea Schubert i Claudio Lovisolo de la Università di Torino. Al Jardí Botànic de Sóller li vull agrair el subministrament de material vegetal. Altra part del material fou donat de manera desinteressada per la Fundació CIPRES del Consell Insular de Mallorca. Per acabar també m’agradaria mostrar la meva admiració a unes quantes persones per la seva tasca no docent ni investigadora. A tots els amics del poble (l’he d’anomenar, Sant Llorenç des Cardassar), autèntics catedràtics del temps lliure, perquè sense ser-ne conscients, entre cerveses, sopars, marxes i sobretot rialles, m’heu sabut desconnectar de tots els mals de cap que duia de Ciutat. A n’Eva no li puc agrair res en concret... sinó que estratègicament col·locada aquí entre línies, ben bé pot fer seus els agraïments anteriors i els que vénen a continuació. I per acabar, i encara que mai seria prou, voldria expressar tota la meva gratitud i admiració a la meva família, i especialment als meus pares, Jeroni i Antònia, que encara que no hagin entès massa bé que és això d’un doctorat, sempre han estat allà, recolzant, mostrant interès, aconsellant, donant coratge i, sobretot, mostrant-se orgullosos del que el seu fill estava fent. ii Symbols and Abbreviations List SYMBOLS AND ABBREVIATIONS LIST α leaf absorptance A anteraxanthin ABA abscisic acid AG gross CO2 assimilation rate AN net CO2 assimilation rate ASAT light- and CO2- saturated photosynthesis ATP adenosine triphosphate β fraction of absorbed light that reaches photosystem II B plant dry matter BL biochemical limitations to photosynthesis C CO2 mole fraction Ca atmospheric CO2 concentration Cc chloroplastic CO2 concentration cDNA complementary deoxyribonucleic acid Chl chlorophyll Ci sub-stomatal CO2 concentration CTAB cetyl trimethyl ammonium bromide ∆F/Fm’ operating quantum efficiency of PSII photochemistry ∆G‡ free energy of activation ‡ ∆Gc carboxylation free energy of activation ‡ ∆Go oxygenation free energy of activation D seed dormancy period DIECA diethyldithiocarbamate DL diffusive limitations to photosynthesis DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate DPS xanthophylls de-epoxidation state DPSMD midday xanthophylls de-epoxidation state DPSPD predawn xanthophylls de-epoxidation state DTT dithiothreitol ε leaf volumetric elastic modulus E leaf transpiration rate EDTA ethylene diamine tetraacetic acid EGTA ethylene glycol tetraacetic acid ES evergreen shrubs ESS evergreen semi-shrubs ETR electron transport rate φCO2 apparent quantum efficiency of CO2 fixation Fm maximum fluorescence in the dark-adapted state Fm’ maximum fluorescence in the light-adapted state Fo basal fluorescence of a dark adapted leaf Fs steady-state fluorescence emission Fv/Fm maximum quantum efficiency of PSII photochemistry Γ CO2 compensation point iii Symbols and Abbreviations List Γ* CO2 compensation point in the absence of mitochondrial respiration G cumulative seed germination gi internal (mesophyll) conductance GRC growth response coefficient gs stomatal conductance gsc stomatal conductance to CO2 gsw stomatal conductance to H2O HEPES hydroxyethyl piperazinyl ethasulfonic acid Jmax electron transport driving regeneration of RuBP Kc Michaelis constant for the carboxylase activity of Rubisco kc rate constant for carboxylation activity of Rubisco cat kc reaction turnover rate for carboxylation activity of Rubisco KL leaf-specific hydraulic conductance Ko Michaelis constant for the oxygenation activity of Rubisco ko rate constant for the oxygenase activity of Rubisco L Rubisco large subunits LA leaf area LAR leaf area ratio LDM leaf dry matter LMR leaf mass ratio MCL mesophyll limitations to photosynthesis NAR net assimilation rate NARp partial net assimilation rate NARt total net assimilation rate NPQ Stern-Volmer non-photochemical quenching of chlorophyll fluorescence NSL non-stomatal limitations to photosynthesis O O2 mole fraction PAR photosynthetically active radiation PCR polymerase chain reaction PEG polyethylene glycol PGA phosphoglycerate PH perennial herbs PMSF phenylmethylsulphonylfluoride PPFD photosynthetic photon flux density Pr photorespiration rate PSI photosystem I PSII photosystem II PVP polyvinylpyrrolidone rbcL Rubisco large subunits encoding genes rbcS Rubisco small subunits encoding genes RD rate of mitochondrial respiration in the dark RDM root dry matter RGR relative growth rate RGRp partial relative growth rate RGRt total relative growth rate RL rate of mitochondrial respiration in the light RMR root mass ratio RNA ribonucleic acid RT reverse transcription reaction Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase iv Symbols and Abbreviations List RuBP ribulose-1,5-biphosphate RWC relative water content RWCMD midday relative water content RWCPD pre-dawn relative water content RWC0 relative water content at zero turgor
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