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…