Iron deficiency stress in algae and cyanobacteria
from global to molecular
Ondrej Prasil IMB Trebon - Algatech
• Iron is the most abundant element of Earth (32%) • Iron is the 4th most abundant in Earth’s crust (5%) • yet today, iron limits primary productivity in 30-60%
of aquatic environments
• Q: why? A: iron concentration and chemistry: Iron aqueous chemistry
In aqueous solutions, Fe has two relevant oxidation states Fe(II) and Fe(III) Ferrous ion (Fe(II)) is relatively soluble and bioavailable at usual pH range (but short lifetime ~10 min at pH 8) Ferric ion (Fe(III)) poorly soluble [0.08-0.2 nM]seawater, easily precipitates -> not bioavailable
Ley, Phycology
Atmosphere composition: orginal archean: CO2, N2, H2O, CO + traces of H2, HCN, H2S, NH3 Nonbiological source of O2: photodissociation of water vapours today atmosphere: 78% N2, 21% O2, 0,035% CO2
Falkowski and Raven, 1998
“Perverse twist of fate” for phototrophs Behrenfeld and Mulligan, 2013
Hohmann-Marriott & Blankenship, 2010
Start of increase of O2 in the atmosphere ~2700-2500 MY Rise of [O2] above 1% between 2100-1900 MY Stage 1 (more than 2.3 GY) both atmosphere and ocean free of O2 Stage 2 (~2.3-1.8 GY) atmosphere oxidized, ocean anoxic and sulfidic Stage 3a (~1.8 – 0.75 GY) deep-ocean euxinia (anoxic H2S rich) Stage 3b (from 0.75 GY to present) both atmosphere and ocean oxygenated
Precipitation of transient metals (Fe, Mn) BIF – Banded Iron Formation hematit Fe2O3 magnetit Fe3O4
pH 0.9-3 (avg 2.3) [Fe] 1.5-20 g/l
Aguilera et al., 2007
Iron is essential micronutrient
Iron is essential micronutrient
Fe/C quota humans: 1Fe / 32,000 C algae and cyanobacteria Fe replete (culture) 1Fe/6,000 C Fe limited eukaryote 1Fe/50,000 C field Fe limited 1Fe/150,000-500,000 C
Iron (unlike other essential trace metals) has higher particulate than dissolved concentration in surface ocean waters. The particulate fraction is biologically inert. >99% of the dissolved Fe(III) [i.e. <0.2 μm] is bound by organic ligands. If not for these ligands, Fe would preciptate and sink. This keeps to retain Fe near the sea surface, where it is reused in photosynthesis. [Fe(OH)2
-] should be <200pM two classes of organic ligands: L1 (stronger) - siderophores L2 (weaker) – cell degradation products together they form “Fe-ligand soup”, concentration is 103 to 105 higher than of Fe(III)
Kranzler et al., 2013
Iron in current aquatic systems
In ocean, Fe ~0.5nM, in surface 70pM, in some freshwater systems, dissolved [Fe] ~ 10 mg/l
Classical (demanding, contamination) Organic extraction (preconcentration and separation from the seasalt matrix) -> graphite furnace AAS Recent Flow injection analysis (FIA), detection chemiluminescence or kinetic spectrophotometry
Measurements of dissolved trace metals
0 50 100 Primary production (gC m-2 month-1)
Productivity of all biosphere = 110 - 120 Gt C year-1
Approx. 50% of productivity on land & 50% in oceans
(annual anthropogenic emisisons 7.1 Gt C)
Hydrothermal vents ~ 0.01 Gt C year-1
Primary productivity of our planet
(Giga = 109)
Behrenfeld and Milligan, 2013
Iron in photosynthesis
3 Fe 6 Fe 14 Fe
Claustre & Maritorena, Science, 2003
What limits primary photosynthetic productivity in aquatic environment? physico-chemical factors: light, temperature, mixing, aerial deposition…. “standard” limiting factors: N,P, (Si for diatoms), light
Iron limitation hypothesis
Iron is essential micronutrient, only source of iron in contemporary ocean is wind-blown dust
Glacial – interglacial changes in dust (=Fe) deposition (up to 50x higher in glacial) -> threefold increase in photosynthesis and drawdown of CO2 to 200 ppm.
“With half a ship load of iron, I could give you an ice age”
John H. Martin
(1935-1993)
High nutrient low chlorophyll regions
Repleted with basic nutrients
NO3-, HPO4
2-
Low phytoplankton
abundance
Eolian iron fluxes
Annually ~ 1011 moles of Fe solubility 1-2%
Aeolian iron supply
Feb 26th 2000.
Sand storm above western Sahara.
SeaWiFS, NASA
April 2010.
Eyjafjallajökull eruption
Iron limitation hypothesis
First bottle experiments
Northeastern Pacific 1980‘s
Southern ocean - SOFeX
January - February 2002
Southern ocean 55° & 66° S, 180° E
R/V Roger Revelle, R/V Melville, R/V Polar Star
SOFeX
South patch evolution
FRR fluorometry
Science 304: 408-414, 2004
Experiments visible from satellites!
A Experiment SOIREE B Crozet C SOFEX
C
Boyd et al., Science, 2007
Mesoscale iron enrichment experiments
Boyd et al., Science, 2007
Mesoscale iron enrichment experiments
Boyd et al., Science, 2007
Limitation of primary productivity by Fe: min. 30% of ocean
Moore et al., 2001
Forms of bioavailable iron
dissolved (<0.2 μm) Fe’- free unchelated pool - readily bioavailable, but at low concentration organically complexed Fe fractions - saccharides for diatoms different siderophores (e.g.ferrated ferroxamine B, aerobactin)- variability in bioavailability decomplexation of siderophores: either reduction of siderophore-bound iron outside of the cells (marine) or import of the Fe-siderophore inside of the cell (freshwater) particulate/colloidal – both organic and anorganic forms
Kranzler et al., 2013
Iron uptake - siderophores Siderophores – the strongest Fe(III) chelators, produced and secreted under Fe starvation Found in >20 species of cyanobacteria (mostly filamentous and heterocystous)
Kranzler et al., 2013
catecholates hydroxamates
Lee, Phycology
For details about synthesis, export and uptake of siderophores in cyanobacteria, see reviews by Kranzler et al. (2013), Morrissey and Bowler (2012)
Siderophores in Anabaena sp.
Siderophores – missing in unicellular marine cyanobacteria (Prochlorococcus, Synechococcus…)
Uptake of free, unchelated, inorganic iron
(reductive iron uptake)
advantageous in dilute environments Km ~ sub nM
Uptake – both reductive or by siderophores costs energy and resources – no free lunch!!
Shuttling of dust particles in Trichodesmium
Rubin et al., 2011
Behrenfeld and Milligan, 2013
Iron in photosynthesis
3 Fe 6 Fe 14 Fe
Physiological responses – lab studies
Iron starvation induces - reduced photosynthetic activity on a pigment basis - changes in organization of the photosynthetic apparatus - oxidative stress (Fenton reactions)
In cyanobacteria, iron limitation decreases rate of synthesis of phycobiliproteins (Fe needed in the synthesis of hemes – precursors of bilins)
Phycobilisomes
Iron starvation induces - concerted decrease in photosynthesis and respiration genes - Photosystem I trimers are monomerized, less effective state transition (psaL depressed) - isiAB operon is upregulated: IsiB (flavodoxin) replaces ferredoxin, IsiA (CP43’) antenna is
highly expressed - idi genes – IdiA protects PSII
IsiA – chla antenna under Fe stress in cyanos
Proposed role of IdiA protection of acceptor side of PSII when phycobilisomes are lacking
Strzepek, Aquafluo meeting, 2007
Effect of Fe on nitrogen fixation
Howard J B , and Rees D C PNAS 2006
7 Fe atoms S 11 Fe
How to detect iron limitation in ocean? sampling (cruises) bioassays molecular or protein markers – flavodoxin/ferredoxin, isiA variable fluorescence remote (satellites) natural fluorescence
Molecular markers for iron limitation Metageomic analysis of light harvesting genes Global Ocean Sampling (GOS) Project (C.Venter) isiA – only at low chl regions
Bibby et al., 2009
BIOSOPE 2004
M.Gorbunov F.Bruyant M.Babin
BIOSOPE
Fe <0.1 nM
N chl
SPG
HNLC HNLC
ENRICHMENT EXPERIMENTS Pumping of surface seawater (30m-depth) (Teflon pump - clean container)
Control
Enrichments in different nutrients
+ Fe
+ Fe
+ NPSi
+ N + FeNPSi
+ FeN
+ dust + FeNP MAR, HNLC
GYR, EGY
METHODOLOGY
4L bottles
3 REPLICATES
2 incubation times: 24h and 48h 50% ambient light
0.0
0.10.2
0.30.4
0.50.6
0.7
MAR HNLC EGY GYR
C
C C C
Fe Fe Fe Fe
Fe 48h
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
MAR HNLC EGY GYR
MAR H
NLC
GYR
EGY
Photochemical quantum efficiency of PSII (Fv/Fm )
C
C C C
Before Fe addition
Fv/F
m
x2.4 x3.3
x1.4
CHLOROPHYLL CONCENTRATIONS (mg.m-3) 48h
0
1
2
3
4
5
MAR HNLC EGY GYR
Normalized values
→ GYR, EGY: no effect of Fe alone
x3
x3.3
→ MAR, HNLC: Fe addition increased Chl a C°
Fe Fe
→ GYR, EGY: N-Fe colimitation for Chl a synthesis
0
1
2
3
4
5
EGY GYR
N
N FeN FeN
x1.5
x4
x2.6 x3
Fe Fe
Behrenfeld & Kolber Science 283, 840
(1998)
050
100150200250300350400450500
20 21 22 23 24 25 26
050
100150200250300350400450500
25 26 27 28 29 30 31
EGY
Transect 1
Fv Fo Fm
Diel cycles in fluorescence yields Fo,
Fm, Fv GYR
EGY
Transect 1
EGY
Transect 1
GYR
EGY
Diel cycles in photochemical yield
Fv/Fm
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
20 21 22 23 24 25 26
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
25 26 27 28 29 30 31
Transect from South Pacific Gyre to Chile: No emission of phycobilisomes in E.Pacific oligotrophic waters
500 550 600 650 700 750 800 85012
1416
1820
22
Wavelength [nm]
Stat
ion
Surface emission spectra
Phycobiliproteins
GYRE EGY
C.Grob et al.
660 665 670 675 680 685 690 695 700
1214
1618
2022
Wavelength [nm]
Stat
ion
Surface emission spectra
HNLC: Shift of emission maximum of PSII 685nm 680nm
EGY
Wavelength [nm]
660 680 700 720
Fluo
resc
ence
[r.u
.]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
685 nm679 nm
Wavelength [nm]
660 680 700 720
680 nm679 nm
Room temperature and low temperature (77K) emission spectra for Gyre and HNLC (EGY) stations
HNLC Gyre
RT RT LT LT
700.9
684.7
678
723.7
650 670 690 710 730
690.7
685
677.3
717.1
650 670 690 710 730
HNLC Gyre
678nm ~ 12% of total PSI + PSII 678nm ~ 22 % of total
Significant increase in the 678 nm peak
650 670 690 710 730 750wavelenght [nm]
678 nm
Additional band 675-678 nm
Emission from uncoupled light harvesting antenna
Fo @ 9 AM [a.u.]0 50 100 150 200 250 300
T C
hla
[mg/
m3]
0.00
0.02
0.04
0.06
0.08
HNLC
GYRE
650 670 690 710 730 750
wavelenght [nm]
Schrader et al., 2011
Distance [km]
0 2000 4000 6000 8000
Fv/F
m
0.0
0.1
0.2
0.3
0.4
0.5
Fv/Fm surface
Gyre
Gyre
HNLC HNLC
HNLC
Distinct biogeochemical regions: ultraoligotrophic center of the South Pacific Gyre: low N, Fe; low biomass, high Fv/Fm HNLC margins: high N, low Fe, low Fv/Fm upwelling of the Humboldt current Claustre et al. (2008), Bonnet et al. (2008)
UPW
UPW
Distance [km]
0 2000 4000 6000 8000
FC
max
0.00
0.02
0.04
0.06
0.08
0.10
Fv/F
m
0.0
0.1
0.2
0.3
0.4
0.5
0.6EQ HNLC1 HNLC1GYRE UPW
Uncoupling between maximum quantum yields: Photosystem II photochemistry (Fv/Fm) and photosynthesis (C fix - Fc
max)
Carbon
Photosystem II
These are not operational, but “potential” or maximal possible yields….
Behrenfeld and Milligan, 2013
Can we detect Fe limitation from space?
Yes! The same phenomena, like in surface fluorescence measurements – high fluorescence yield/chl (ϕsat)
Behrenfeld et al., 2009
Thank you for your attention…