Pyridine nucleotide levels in zooplankton: laboratory work and field samples

Py idine nucleo ide le els in zooplank on: labo a o y and ield samples Ciclo de Ciencia Compa ida, 12 Junio 2012 Osma N., Packa d T. and Gómez M. Ins i u e o Oceanog aphy and Global Change, Biological Oceanog aphy G oup, Uni e si y o Las Palmas de G an Cana ia, Cana y Islands, Spain. E-mail add ess: nosma@beca ios.ulpgc.es Respi a ion - Ubiqui ous p ocess - Key componen in he es ima ion o he ca bon lux. Ne e heless… ¿¿OCEANIC RESPIRATION ?? Del Gio gio and Dua e, 2002 Due o - Di icul o quan i y in si u espi a ion a es. - Di ec measu emen s  Incuba ions SOLUTION!! Biochemical assays Po en ial Respi a ion (F) Elec on anspo sys em (ETS) ac i i y o es ima e espi a o y oxygen consump ion (RO2) Packa d e al., 1971 Vmax Subs a e sa u a ion Wha mechanism con ols he RO2? Physiological RO2 ETS ac i i y Packa d e al., 1996 Ho: In i o po en ial espi a ion de e mined by he Vmax and he subs a es a ailabili y . RESPIRATION MODEL BASED ON SUBSTRATE LIMITATION Py idine nucleo ides and succina e Py idine Nucleo ides (PNs) NAD NADP (Be ge e al., 2004; Ziegle , 2005; Pollack e al., 2007; Ying, 2008) NAD+ NADH Ca abolism NADPH NADP+ Anabolism  Reducing equi alen s o hund ed o cellula eac ions.  Func ions: - Modula ing ene gy me abolism, educ i e biosyn hesis and an ioxida ion p ocesses. - Recen s udies, impo ance in cell signaling in animals, plan s and ungi. P e ious Wo k  Few s udies in ma ine o ganism Animal physiology - Du ing 50’s and 60’s, de elopmen o di e en echniques o measu e PNs (Chance e al.,1955,1962; Low y e al. 1961). - Recen clinical applica ions (Maye sky and Chance, 2007). Plan physiology - Knowledge inc easing s eadily  Le els and oles unde di e en me abolic condi ions. (Molle and Rasmusson, 1994; Hage don, 2004; Noc o e al., 2006) Bac e ial ac i i y - Wos and Polla d (2009): NADH as an index o bac e ial me abolic ac i i y in ac i a ed sludge. - NADP+ in ela ion o isoci a e dehyd ogenase ac i i y (Roy and Packa d, 1998). - A emp ed o es ima e ma ine p ima y p oduc ion wi h he NADPH (S eigenbe ge e al., 2004), . P e ious Wo k  Few s udies in ma ine o ganism Animal physiology - Du ing 50’s and 60’s, de elopmen o di e en echniques o measu e PNs (Chance e al.,1955,1962; Low y e al. 1961). - Recen clinical applica ions (Maye sky and Chance, 2007). Plan physiology - Knowledge inc easing s eadily  Le els and oles unde di e en me abolic condi ions. (Molle and Rasmusson, 1994; Hage don, 2004; Noc o e al., 2006) Bac e ial ac i i y - Wos and Polla d (2009): NADH as an index o bac e ial me abolic ac i i y in ac i a ed sludge. - NADP+ in ela ion o isoci a e dehyd ogenase ac i i y (Roy and Packa d, 1998). - A emp ed o es ima e ma ine p ima y p oduc ion wi h he NADPH (S eigenbe ge e al., 2004), . Up o now, NO wo k in ma ine o ganisms Desc ibing in acellula le els o PNs Compa ing PNs le els wi h espi a ion a es Labo a o y Wo k Oxy his ma ina He e o ophic dino lagella e. High ole ance o change in empe a u e, salini y and nu ien s  Good o cul u ing! Why??  Easy o cul u e  Model o ganism  espi a o y me abolism?? D oop,1959; Kimmance e al., 2006; Jeong e al., 2003 Oxy his ma ina, A is izabal (2009) 20 µm Rhodomonas salina  Fed wi h Rhodomonas salina  Same cul u ing condi ions, excep o ligh Field Samples C uise ack o he R/V Hespe ides du ing he LEG 3, 4 and 7. •LEG 3,4: Feb ua y - Ap il 2011. •LEG 7: June - July 2011. Sampling Me hods SIZE FRACTIONATION 150 - 200 m sampling 100 – 500 µm 500 – 1000 µm > 1000 µm 100 µm mesh size WP-2 ne 0 h: Well ed 24 h: S a ed RESPIRATION MEASUREMENTS ON BOARD S o age a - 80oC LABORATORY WORK AT INSTITUTE ETS ACTIVITY INTRACELLULAR SUBSTRATES PROTEIN MASS Resul s & Discussion Speci ic espi a o y oxygen consump ion dec eases wi h size ac ion, bo h in well- ed and s a a ion condi ions. No signi ican ly di e ences o he R/F a io be ween well- ed and s a ed o ganisms. Resul s & Discussion 100-500 µm 500-1000 µm > 1000 µm Speci ic NADH and NADPH concen a ion no signi ican ly di e en in he h ee size classes. No signi ican ly di e ences be ween well- ed and s a ed o ganisms, - Excep ion: NADH le els in 100-500 m size ac ion Conclussions Conclussions The all in he espi a ion du ing he onse o s a a ion sugges s ha espi a ion is subs a e limi ed du ing his pe iod. To al PNs and espi a ion a e well co ela ed du ing s a a ion in a ma ine dino lagella e. This obse a ions suppo s he use o a espi a ion model based on subs a e limi a ion. Labo a o y wo k … ENZYME KINETIC MODEL Ro = Vmax: Maximum eloci y o he eac ion S1, S2: Subs a e concen a ion o NADH and NADPH K1, K2: Michaelis cons an o NADH and NADPH Kia: Appa en dissocia ion cons an 𝑉𝑚𝑎𝑥 · [𝑆1 ·𝑆2] 𝐾1 · 𝐾𝑖𝑎 + 𝐾2 · [𝑆1] + 𝐾1 · [𝑆2] + [𝑆1 ·𝑆2] Packa d and Gomez, 2008 Field da a … E ec o s a a ion seen in 100-500 µm size class. Highes size classes migh ha e a la ge ese e and 24 h o s a a ion migh no challenge hem. Acknowledgemen s This esea ch is amed in he EXZOME p ojec (CTM 2008 – 01616/MAR), which is unded by he ex inc Spanish Science and Educa ion Minis y. N. Osma ecei es inancial suppo om he Fo ma ion and Pe ec ion o he Resea che Pe sonal P og am om he Basque Go e nmen . I also hank o MALASPINA 2010 (CSD-20080077) p ojec o in i ing me o pa icipa e in i s c uise. Py idine nucleo ide le els in zooplank on: labo a o y and ield samples Ciclo de Ciencia Compa ida, 12 Junio 2012 Osma N., Packa d T. and Gómez M. Ins i u e o Oceanog aphy and Global Change, Biological Oceanog aphy G oup, Uni e si y o Las Palmas de G an Cana ia, Cana y Islands, Spain. E-mail add ess: nosma@beca ios.ulpgc.es