A functional gene approach to studying nitrogen cycling in the sea. Matthew Church (MSB 612 / March 20, 2007

Transcription

1 A functional gene approach to studying nitrogen cycling in the sea Matthew Church (MSB 612 / mjchurch@hawaii.edu) March 20, 2007

2 Overview Climate change, carbon cycling, and ocean biology Distributions of nutrients and marine production The ocean s nitrogen cycle Case Study: Nitrogen fixation from a functional gene perspective

3 CO 2 concentrations in the atmosphere-past and present

4 Time Scales of Carbon Exchange in the Biosphere

5 380 pco The Ocean s response to increasing CO 2 : Temporal variability in surface ocean CO 2 and ph pco 2 (µatm) ph (total scale, in situ T) Air Sea ph

6 In the open ocean, biology controls bioelemental cycling

7 The Influence of Biology on Carbon Cycling Depth (m) Total inorganic carbon (µmol C kg -1 )

8 Temperature What controls the distribution of marine biological production? Ocean primary production Nitrate Mike Behrenfeld,

9 Depth profiles of light, temperature, nutrients, and chlorophyll in the open ocean 0 Temperature ( o C) NO NO 2 - (µmol L -1 ) Depth (m) Irradiance (mmol quanta m -2 s -1 ) Chl a (µg Chl a L -1 )

10 Satellite-derived estimates of global chlorophyll distributions

11 Ecosystem controls on marine production Physical: turbulence, light, temperature Chemical: nutrient availability, trace elements Biological: community composition, food web structure, N 2 -fixation Climate and Human Influences: ENSO, PDO-NAO, land use, population, deserts-dust

12 OLIGOTROPHIC GYRES OF THE WORLD OCEAN Basin Pacific Atlantic Indian Other TOTAL Area (x 10 3 km 2 ) 90,105 30,624 19,599 8, ,329 %

13 The Hawaii Ocean Time-series (HOT) Station ALOHA Established in 1988 as part of the U.S. JGOFS program. Monthly measurements of ocean physics, biology, and chemistry at Station ALOHA Primary objectives: characterize timedependent dynamics in carbon, nitrogen, and phosphorus inventories and fluxes.

14 01/04 Highly stratified ocean ecosystems =high light, very low nutrient concentrations = low chlorophyll 0 MLD 1% PAR Depth (m) /98 07/98 01/99 07/99 01/00 07/00 01/01 07/01 01/02 07/02 01/03 07/03 Sampling data

15 Vertical profile of light, nutrients, and primary production NO NO 2 - (nmol L -1 ) Depth (m) PP Chl a MLD 1% PAR NO 3- + NO Chl a (µg Chl L -1 ) 14 C-Primary production (µmol C L -1 d -1 ) [ NO 3- + NO 2- ] ~ 1-10 nm [ PO 4 3- ] ~ nm Annual rates of net primary production >15 mol C m -2

16 Global Models and Predictions Ocean circulation models coupled with biology Increased temperature will impact both CO 2 the oceans ability to absorb atmospheric CO 2

17 In the low nutrient upper ocean very small, but very diverse plankton populations comprise the majority of biomass and production

18 The microbial soup at Station ALOHA µm µm Photosynthetic unicellular bacteria (Prochlorococcus, Synechococcus) Colonial cyanobacteria bacteria (Trichodesmium) µm Non-photosynthetic Bacteria and Archaea

19 New production in the ocean 2 forms of primary production: 1) new production supported by external input of N (e.g. NO 3- and N 2 ), 2) recycled or regenerated production, sustained by in situ recycling of N. -Over short time scales, input of new N supports higher trophic levels and supports carbon export.

20 The ocean nitrogen cycle Nitrogen serves as both an essential nutrient, and because of its redox potential also serves as an energy source and electron acceptor. Denitrification: NO - 3 NO - 2 NO N 2 O N 2 Nitrification: NH + 4 NO - 2 NO - 3 Nitrogen fixation: N 2 NH + 4 Anammox: NO 2 + NH + 4 N 2 + 2H 2 O

21 N 2 fixation contributes a major fraction (~30-84%) of new production in the open ocean Dore et al. (2004)

22 Nitrogen sources supporting plankton growth Eukaryotes Prochlorococcus Eukaryotes Diazotrophs Heterotrophs 10 µm 10 µm 100 µm 10 µm NO 3 - NO 2 - NH 4 + N 2 (*) NO 2 - NH 4 + DON NO 3 - NH 4 + N 2 NO 3 - NO 2 - NH 4 + DON N 2 Not all nutrients are created equal

23 Genes and processes in the nitrogen cycle nirk nirs NO 3 narb NO 2 Nitrate assimilation Denitrification norb N 2 O NO nira hao NH 2 OH Ammonia oxidation nosz aax N 2 nifh N 2 fixation NH 4 + glna DON amoa Ammonium assimilation Identifying genes encoding enzymes that catalyze specific biochemical reactions

24 Selected sources of nitrogen supporting plankton growth in the upper ocean N 2 nitrogenase (nif) nitrate / nitrite reductase (nar, nir) glutamine synthetase (gln) Urease (ure) DON NO 3 - NO 2 - NH 4 +

25 Nitrogenase Fe protein MoFe protein Fe protein nifh nifh nifdk Fe protein is inactivated upon oxidation; cyanobacteria must isolate nitrogenase (spatially or temporally) from O 2 evolved during photosynthesis.

26 From genes to proteins nifh nifd nifk nif operon 5 -TATTAGGCCAATCCGC-3 3 -ATAATCCGGTTAGGCG-5 nifh gene Transcription of nifh DNA Translation of nifh mrna 5 -UAUUAGGCCAAUGGCG-3 nifh mrna transcript Nitrogenase

27 Phylogenetic relationships among nifh genes Cluster IV Cluster III Cluster II 0.10 Cluster I

28 1. What are the predominant N 2 -fixing bacteria in the open ocean?

29 Methodological approaches Sample and extract plankton DNA and mrna. Amplify nifh genes by PCR and reverse transcriptase (RT) PCR using degenerate primers, clone and sequence amplified products T C C A C A C G T C T T A T T C C G C A C T C T C C A C A C G T C T T A T T C T G C A C T C T C T A C T C G T T T A A T C T T AAA C G C T T C T A C A C G T T T G A T G C T G C A C T C T T C T A C A C G T T T G A T G C T G C A C C C T T C T A C T C g T T T A A T G T T G C A C T C T C T A C T C G T T T A A T G T T G C A C T C

30 100 µm Diazotrophic bacteria at Station ALOHA Richelia Heterocystous cyanobacteria 100 µm 2-10 µm µm Unicellular cyanobacteria Group A and B Filamentous cyanobacteria Trichodesmium >0.2 µm >0.2 µm Proteobacteria?? Anaerobes

31 Questions 1. How abundant are nifh-containing microorganisms? 2. What processes regulate nifh expression and N 2 fixation?

32 An introduction to the quantitative polymerase chain reaction (QPCR) Environmental DNA or reverse transcribed mrna Denature Anneal Extend 94 o C 64 o C Forward primer Probe Reverse Primer Fluorescence is directly proportional to the abundance of the initial target i.e., C n = C 0 * 2 n Where n=pcr cyles

33 Vertical distributions of various nifh phylotypes at Station ALOHA (Dec. 2002) Group B Group A Cluster III Trichodesmium spp MLD Depth (m) 100 1% PAR <10 µm >10 µm nifh genes (copies L - 1 ) nifh genes (copies L - 1 )

34 Temporal patterns of nifh expression at Station ALOHA (Dec. 2002) Group A unicellular Group B unicellular 25 m Nitrogenase Expression (nifh transcripts L -1 ) Nitrogenase Expression (nifh transcripts L -1 ) Trichodesmium spp. Heterocystous cyanobacteria 25 m 00:00 06:00 12:00 18:00 00:00 06:00 12:00 18:00 00:00 06:00 12:00 Dec. 13, 2002 Dec. 14, 2002 Dec. 15, 2002