John A Raven - PIK Research Portal · • Conclusion that biochemical constraints only restrict the...

Redfield through the ages

John A RavenDivision of Plant Sciences

University of Dundee at the James Hutton InstituteInvergowrie, Dundee DD2 5DA,

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• The Redfield Ratio today• Biogeochemical changes related to the

Great Oxygenation Event• Possible implications for Early Proterozoic

Elementary Stoichiometry• Conclusions

The Redfield Ratio Today

• The Redfield Ratio refers to the ratio of the elments C, H and N in marine planktonicprimary produces (= marine phytoplankton)

• By atoms, the ratio is C106: N16: P1• Redfield 1934 James Johnstone Memorial Volume (R.J. Daniel

editor) pp.176-192. University of Liverpool.• Redfield 1958 American Scientist 46 205-221


• The Redfield Ratio was originally determined from the elemental content of phytoplankton and the variations in C, N and P above and below the permanent thermocline

• Represents a spatial and temporal average of the elemental composition of marine phytoplankton

The Redfield Ratio Today• ‘Extended Redfield Ratio’ includes other

elements such as Fe:

• C106:N16:P1:Fe0.01• 29 phylogenetically diverse species of

Cyanobacteria and Algae grown in nutrient replete conditions had a mean elemental ratio of

• C132:N18:P1:Fe0.018• Quigg, Irwin and Finkel 2011 Proc Roy Soc B 278 526-534

The Redfield Ratio Today• There is significant phenotypic

variation in the C:N:P:Fe ratios in marine phytoplankton as a function of the ratio of elements in an available form in the growth medium

• Conclusion that biochemical constraints only restrict the N:P ratio to between 10 and 50

• Geider and La Roche 2002 Journal of Phycology 37: 1-17

The Redfield Ratio Today• Much of the ocean is N-limited, as shown

by enrichment experiments with ‘combined’ N (biologically available N other than N2)

• Large resource costs of diazotrophicreplacement of the available N lost in denitrification?

• ‘Explains’ why P, the predicted biogeochemically limiting element, does not limit productivity based on combined N in much of the ocean, but does (with Fe) limit diazotrophy


• Several attempts have been made to give a mechanistic basis to the Redfield Ratio

• A recent model is based on the Growth Rate Hypothesis for the increasing N:P ratio in organisms as a function of increasing growth rate

• N:P a surrogate for protein:RNA ratio• Loladze and Elser 2011 Ecology Letters 14: 244-250

The Redfield Ratio Today• What can studies on the Redfield Ratio

today tell us about what might have happened in the past?

• Geider and La Roche (2002) say “None of our analyses have suggested that the average elemental composition of phytoplankton is fixed, or should have remained invariant over past geological time”


• What can studies on the Redfield Ratio today tell us about what might have happened in the past?

• The Loladze and Elser (2011) model could be run in the past to see how different environmental conditions could change the inputs

Biogeochemical Changes related to the Great Oxygenation Event

• Increased O2 in the biosphere from oxygenic photosynthesis began 2.2 – 2.4 Ga ago

• Earliest biomarker evidence of cyanobacteria 2.15 Ga ago, eukaryotes (not necessarily photosynthetic) 1.78 Gaago (Rasmussen et al. 2008 Nature 455: 1101-1104)

• Earliest body fossil evidence for eukaryotic algae at least 1.2 Ga ago

• Lane 2004 Oxygen. The Molecule that made the World. OUP

History of the Earth's oxygenation, showing important constraints described in the text.

Kirschvink J L , Kopp R E Phil. Trans. R. Soc. B 2008;363:2755-2765

©2008 by The Royal Society

Bangiomorpha, a multicellular red alga (Rhodophyta: Bangiophycese) from rocks at

least 1.2 Ga old


• Presumably oxygenic photosynthesis in cyanobacteria was the source of the O2 in the biosphere from about (2.2-) 2.4 Gaago

• Cyanobacteria must have occurred several 100 Ma before the earliest biomarkers of them from 2.15 Ga old rocks


• Global oxygenation required marine photosynthesis: freshwater habitats are, and were quantitatively, inadequate

• Molecular phylogeny shows living relatives of basal cyanobacteria live in freshwater, so ‘escape’ to the ocean must have preceded global oxygenation

• Blank and Sánchez-Baracaldo 2010 Geobiology 8: 1-23

Biogeochemical changes related to Global Oxygenation

• O2 build-up needs:• Oxygenic photosynthesis• In addition, O2 accumulation in the

biosphere requires that the rate of organic carbon burial (removing the organic carbon from respiratory oxidation with consumption of O2) exceeds the rate of O2 consumption in oxidation of Fe2+ → Fe3+,

Mn2+ →Mn4+ and S2-→ SO42-


• O2-consuming respiration became globally significant

• Production of reactive oxygen species (ROS) as a result of redox reactions in the presence of O2 : 1O2, O2

•-, H2O2, OH•

• Attenuation of UV-B by stratospheric O3, so less damage from direct absorption of UV-B by nucleic acids, proteins, quinones, etc. and by production of ROS


• Greenhouse effect of O3

• Photodamage to PSII (the O2-producing photosystem) is partly O2-dependent

• Suggestion 10 years ago that photosynthetic O2 production was subject to thermodynamic feedback inhibition by O2 within the ecological range of O2 concentrations has now been disproved

• Raven and Larkum 2007 Photosynthesis Research 94: 31-42• Shevela et al 2011 PNAS doi: 10.1073/pnas.104249108


• Allows nitrification but can be exceeded in hypoxic and anoxic zones by denitrification

• Critical concentration of O2 above which nitrification exceeds denitrification, allowing NO3

-

to be a stable component of the photic zone• O2 damages nitrogenase – decreased

production of combined nitrogen and increased resource cost of diazotrophy

• Falkowski & Godfrey 2008 Phil Trans RS B 363: 2705-2716; Raven JA (2011) Physiologia Plantarum 147: 87-104.


• O2 compete with CO2 at active site of Rubisco –needs more energy to fix CO2

• More protein needed to make more Rubiscoplus carbon oxidation cycle and/or carbon concentrating mechanisms

• Decreased photosynthetic rate per unit biomass with increased resource cost

• Five other autotrophic CO2 fixation pathways: all show complete or partial inhibition by O2

• Berg 2011 Appl Env Microbiol 77: 1925-1936; Raven JA (2011) Physiologia Plantarum 147: 87-104


• Resynthesis of proteins damaged in photoinhibition and O2 damage to nitrogenase needs addition RNA, hence extra P

• Synthesis of additional proteins related to O2 inhibition of Rubisco needs additional RNA, hence extra P

• P less available because bound to Fe3+

• Fe3+ less available than Fe2+

• Raven JA (2011) Physiologia Plantarum 147: 87-104.


• Decreased Fe availability in oxygenated habitats

• Increased need for Fe in nitrogenase(diazotrophy favoured by loss of combined N in nitrification – denitrification cycles)

• Increased need for Fe in NO3- reductase

and, especially, NO2- reductase as NO3

-

rather than reduced N predominates in some phytoplankton habitats

Possible implications for Early Proterozoic Elementary Stoichiometry

• Decreased availability of N, P and Fe• Potential for greater need for each of

these elements per unit biomass as a result of the Oxygenation Event, assuming an approximation to optimal allocation

• What might this do to the Redfield Ratio, assuming that this is in some way related under all resource supply conditions to the biochemistry of the organism?


• Focus on P: impact of oxygenation has been less commonly considered than has N and Fe

• Obvious impact of oxygenation is on through damage to proteins, and especially proteins in photosystem II and in nitrogenase

• Breakdown of damaged proteins and synthesis of replacements has little implication on net N requirement for growth

• Could have greater impact on net P requirement for growth via need for more P-rich RNA (especially rRNA) to make more protein as a result of damage to protein


gC/gdw gN/gdw gP/gdwProtein 0.53 0.16 <0.001RNA 0.34 0.16 0.091Ribosome 0.44 0.16 0.046Geider and La Roche 2002 European Journal of Phycology 37: 1-17

Atom C/Atom P Atom N/Atom P Atom P/Atom PProtein >1770 >460 1RNA 9.3 3.7 1Ribosome 24 7.3 1Redfield 106 16 1Assuming an upper limit of 1 P per mean 45000 Mr of protein

P pools in cyanobacteria and algae

• Most measurements of P pools cyanobacteriaand algae are pre-2000 (metabolomicsnotwithstanding)

• Similar ratios of P pools (other than storage pools) in cyanobacteria, algae and plants

• Storage pools in cyanobacteria and algae: vacuolar orthophosphate; polyphosphate bodies

• Storage pools in plants: vacuolarorthophosphate, phytic acid

• Bieleski 1973 Annual Review of Plant Physiology 24: 225-241;Kanai & Simonis 1968 Archiv fur Mikrobiologie 63: 29-40

Phosphate fractions other than inorganic P, poly-P and phytic acid

P Fraction relativeTo Ester P = 1

Young photosynthetic tissue of flowering plant

Unicellular green alga

DNA 0.2 0.2RNA 2 2.5Lipid P 1.5 1.7Ester P 1 1

P allocation to RNA

• RNA: most RNA is rRNA. Some cyanobacteriaand algae apparently have ‘excess’ rRNArelative to what is needed for net protein synthesis in growth plus ‘normal’ turnover, especially when growth is not limited by P

• Flynn et al. 2010 Journal of Phycology 46: 1-12

• Fits with optimal allocation rather better than with growth rate hypothesis

• Part of ‘excess’ rRNA could used to repair damage from O2 effects to certain proteins

Possible problems with removing part of the ‘non-storage’ P fractions

• Excess rRNA could be needed in repair of damage by O2 of nitrogenase in diazotrophsand/or in mechanisms which restrict this damage, and in repair of photodamage to the D1 protein of PsII

• Example is the marine diazotrophiccyanobacterium Trichodesmium, using compositional data on material from the Gulf of Mexico

• Brown et al. 2008 Marine Biology 154: 413-422

Trichodesmium coloniesMaximum dimension 1 – 2 mm

Trichodesmium filaments in a colonyFilaments about 10 μm diameter

P allocation to RNA

• Nitrogen fixation in Trichodesmium involves a breakdown and resynthesis of nitrogenase once per diel cycle

• Nitrogenase is 3.8% of total protein, relative growth rate is 0.3 per day, so complete nitrogenase turnover each day involves synthesis of an additional 3 units of nitrogenasein addition to the 1 unit accounted for in net protein synthesis, i.e. 3 x 3.8% or a 11.4% increment of protein synthesis over that for net protein synthesis.

P allocation to RNA

• O2 damage to nitrogenase involving breakdown and resynthesis equivalent to another turnover of the complete nitrogenase complement each day requires another 3 times 3.8% (= 11.4%) of protein synthesis above the baseline value, a total of 23% extra

• Diazotrophy in Trichodesmium could involve 1.23 times the ribosomal catalytic capacity predicted from the net rate of protein synthesis

• 1.12 times the non-storage P quota

P allocation to RNA

• The marine cyanobacterial diazotrophCrocosphaera switches Fe between photosynthetic apparatus and nitrogenasein each diel cycle (decreases cell quota of Fe compared to having both the major Fe-using pathways active at the same time) and has a similar growth rate to that of Trichodesmium

• Saito et al. 2011 PNAS doi:10.1073.1006943108

P allocation to RNA

• May not need additional ribosomes to replace photosynthetic apparatus and diazotrophic apparatus each diel cycle: same set of ribosomes used the two, temporally separated, protein synthetic process

P allocation to RNA

• Increased P requirement for ribosomesrelated to resynthesis of O2-damaed nitrogenase could help account for higher P demand for diazotrophy than for use of combined N

• Contribution to relative rarity of diazotrophy?

P allocation to RNA

• Photodamage to Photosystem II: D1 protein is only about 0.1% of total cell protein in Trichodesmium, so even with more rapid damage, and hence resynthesis, than for nitrogenase there is a smaller need for additional ribosomes

• There is also photodamage to other proteins in Photosytem II

Comparison of effects on P quota with replacement of P-lipids

• Lipid P replaced by S-lipid and galactolipidin thylakoids of oxygenic photolithotrophs: little S-lipid in anoxygenic photosynthetic bacteria

• Additional S-lipid (and galactolipid) associated with oxygenic photochemistry and hence with the Oxygenation Event

Comparison of effects on P quota with replacement of P-lipids

• Lipid P: Replacement of P-lipid with S-lipid (S-lipid already dominant charged lipid in thylakoids) may have functional implications

• If S-lipid gave the same fitness as P-lipid, why has not P-lipid been universally replaced by S-lipid, and not just in very P-deficient but S-sufficient habitats, such as the oceanic gyres: external S:P ≥ 107?

• Van Mooy et al. 2009 Nature 458: 69-72

Comparison of effects on P quota of replacement of P-lipids

• Replace 90% of P-lipid with S-lipid, as found with some extant marine phytoplankton

• Decreases the cell quota of non-storage P to 0.72 of the quota with the ‘normal’ P-lipid content.

• Compare with 1.23 times cell P for diazotrophy in Trichodesmium

Observed P use efficiencies

• Phosphorus use efficiencies in growth (biomass produced per unit cell P per unit time) can be calculated from literature data

• No attempt made to correct for subsistence P quota: value unknown in most cases

• Used data for highest internal P value which is non-saturating for growth minimizes the impact of the subsistence quota

Phosphorus Use Efficiency (PUE) of Cyanobacteria:

gdw increase· (mol cell P)-1 ·d-1

Cyanobacterium PUE ReferencesAphanizomenon (N = NH4

+)3125 De Nobel et al.

(1998)Aphanizomenon (N = N2)

2678 De Nobel et al. (1998)

Prochlorococcus Med 4 1413 Bertilsson et al. (2003)

Synechococcus WH 8012

2257 Bertilsson et al. (2003)

Synechococcus WH 8103

2016 Bertilsson et al. (2003)

Synechococcus 1600 Mand & Brown (1981)

Phosphorus Use Efficiency (PUE) of Chlorophyta (C) and Rhodophyta (R):

gdw increase· (mol cell P)-1 ·d-1

Phylum, Class Alga PUE ReferenceC, Charophyceae Cosmarium abbreviatum 1099 Spijkerman & Coesel

(1996)

C, Charophyceae Staurastrum pingue 480 Spijkerman & Coesel(1996)

C, Charophyceae Staurastrum chaetoceros 410 Spijkerman & Coesel(1996)

C, Chlorophyceae Scenedesmus quadricauda 1500 Lang & Brown (1981)

R, Bangiophyceae Porphyra yezoensisHigh PAR, N = NO3

- 1378 Hofling (1999)

R, Bangiophyceae Porphyra yezoensisLow PAR, N = NO3

- 861 Hofling (1999)

R, Bangiophyceae Porphyra yezoensisHigh PAR, N = NH4

+ 417 Hofling (1999)

R, Bangiophyceae Porphyra yezoensisHigh PAR, N = NH4

+ 233 Hofling (1999)

Staurastrum seen from end (L) and side (R)About 40 μm across

Cosmarium: two pairs of semi-cells about to divideSemi-cells about 30 μm wide


• Significant inter-specific variation in PUE for growth under as near ‘optimal’conditions as were used in the various data sets

• Probably some genotypic effects as well as influence of different conditions (e.g. temperature) among data sets


• Data are consistent with additional P requirement for ribosomes (= lower PUE) for diazotrophy rather than growth on combined N for Aphanizomenon

• Data are not consistent with need for extra P in ribosomes for repair of photodamageto the D1 protein in Porphyra grown at high light; growth light-limited at low light

Aphanizomenon flos-aquaeOval, almost transparent, heterocysts fix N2; elongate resting spores

(akinetes); other cells photosynthesise

PorphyraThalli 100 mmacross

Additional Data Needed• Comparison of protein turnover rates,

protein:RNA ratios and phosphorus use efficiencies for growth of:

• Anoxygenic purple photosynthetic bacteria growing photosynthetically in the absence of oxygen and aerobically in the dark on organic substrates

• Yeast aerobically and anaerobically(fermentatively) on glucose

Conclusions

• Major changes to biogeochemical cycles with the Oxygenation Event

• Previous studies concentrated on C, N and Fe cycles

• Possible effects of oxygenation on non-storage P requirements is a variation from about -30% to about +23%

• More data needed

Acknowledgements

• Susan Allen, Mitchell Andrews, John Beardall, Birgitta Bergman, Mark Brundrett, Paul Falkowski, Zoe Finkel, Kevin Flynn, Mario Giordano, John Hodgson, Steve Hopper, Andy Knoll, Hans Lambers, Stephen Maberly, AntoniettaQuigg, Simon Roberts, Sally Smith, KayleyUsher

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