Nitrogen Fixation and Nitrogen Fixation and Regulation of Photosynthesis for Nitrogen FixationRegulation of Photosynthesis for Nitrogen Fixation

Hendrik Küpper, Advanced Course on Bioinorganic Chemistry & Biophysics of Plants, summer semester 2013

Part I: Part I: Nitrogen fixationNitrogen fixationNitrogen fixationNitrogen fixation

Nitrogen cycle (I): biological processes

Im Wasser An Land

von: www.oceanography.geol.ucsb.edu von: www.hypersoil.uni-muenster.de

Nitrogen cycle (II): intermediate steps

NO3-

Nitrite oxidaseNitrite oxidase Nitrate reductaseNitrate reductase

NONO2

-

Anammox N2OH NOH Anammox N2OH2NOH

N2

NH3

Organisms involved in nitrogen fixation

Anabaena TrichodesmiumRhizobienAzolla

AzotobacterGloeothece

Total equation of biological nitrogen fixation

N2 + 8H+ + 16 MgATP + 8e-N2 + 8H + 16 MgATP + 8e

2NH3 + H2 + 16 MgADP + 16 Pi

Mechanism of biological nitrogen fixation:nitrogenase of cyanobacterianitrogenase of cyanobacteria

The iron protein

Nitrogenase: P-Cluster

Nitrogenase: M-Cluster

Structure of Mayer and Smith, 1999 1 6 Å resolution: discovery1999, 1.6 Å resolution: discovery of the "central nitrogen", meanwhile re-identified as central carbon (Spatzal Thomas; Aksoyoglu Muege; Zhang Limei; t l 2011 S i 334et al. 2011, Science 334;

Lancaster Kyle M.; Roemelt Michael; Ettenhuber Patrick; et al., 2011, Science 334)

Nitrogenase: Mechanism of nitrogen reduction

Nitrogenase: putative intermediates of nitrogen reduction

Barney B_et al (2006) Breaking the N2 triple bond- insights into the nitrogenase mechanism. DaltonTransactions 2277-84

Nitrogenase: central C

Spatzal T et al et Einsle O (2011) Science334, 940- Lancaster KM_et al et Neese F et DeBeer S (2011) Science334_974-

Nitrogenase: putative intermediates of nitrogen reduction

Part II:Part II:Regulation of photosynthesis for nitrogen fixationRegulation of photosynthesis for nitrogen fixation

Evolution of biological nitrogen fixation in comparison to photosynthesis

Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_157-164

Strategies of photosynthesis regulation for nitrogen fixation in cyanobacteriay

Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_157-164

Unicellular cyanobacteria

Regulation of photosynthesis for nitrogen fixation in unicellular cyanobacteria (II)y ( )

glycogen storage begins

light period: carbon + energy storage

max. photosynthetic capacitydown regulation of photosynthesiscarbon energy storage photosynthesis

maximum glycogen storage

nitrogen fixation beginsup regulation ofphotosynthesis

dark period: nitrogen fixation

y

maximum nitrogen fixationmaximum respiration

Heterocyst forming cyanobacteria

vegetative Zellen

Pro-Heterocyste

Heterocyste

From: Culture service of Instituto de Bioquímica Vegetal y Fotosíntesis, Sevilla, Spain

From:El-Shehawy et al 2003 Physiol Plant 119 (1), 49-55

Heterocyst differentiation:distribution maps of chlorophyll fluorescence kinetic

M i l fl t i ld (F )

distribution maps of chlorophyll fluorescence kinetic parameters

Maximal fluorescence quantum yield (Fm)

25 µm

13 h 36 h 55 h 112 h

Photosystem II activity (F /F )Photosystem II activity (Fv/Fm)

Ferimazova N, Setlík I, Küpper H, unpublished

Heterocyst differentiation:changes in parameters of chlorophyll fluorescence kineticschanges in parameters of chlorophyll fluorescence kinetics

Ferimazova N, Setlík I, Küpper H, unpublished

Trichodesmium:anoxygenic photosynthesis energizing nitrogen fixation

i th ll d i th h t i din the same cells during the photoperiod

Trichodesmium• Marine filamentous, non-heterocystous cyanobacteria• contribution of Trichodesmium to marine N2 fixation: 30-50%• Nitrogen fixation is confined to the photoperiod and occurs

simultaneously with oxygenic photosynthesissimultaneously with oxygenic photosynthesis. • How nitrogenase is protected from damage by

photosynthetically produced O2 has remained an enigma.photosynthetically produced O2 has remained an enigma.

Surface blooms in the Colonies – tuft and puffSurface blooms in the Arafura Sea

Colonies tuft and puff formation

)351 1Aktivitätszyklus von Trichodesmium

Trichodesmium

n fix

atio

n

µg c

hl a

-1 h

-1

25

30

35

e F v

/Fm

0.9

1.0

1.1

T i h d i

Nitr

ogen

nmol

C2H

2 µ

10

15

20

Rel

ativ

e

0.7

0.8Trichodesmium-

bloom

(n

0

5

0.5

0.6

h-1) 60

1 )

-2

2 µ

g ch

l a-1

h

30

40

50

resp

irat

ion

µg c

hl a

-1 h

-1

-10

-8

-6

-4

GP

(µM

O2

0

10

20

Dar

k r

(µM

O2

µ

-16

-14

-12

10

colonies: “Tuft” and “Puff”

Local time (h)

8 10 12 14 16 18 20 22-20

-10

-20

-18

( )

Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294, 1534-1537

Co-Localisation of nitrogenase and PSII in Trichodesmium

D1 protein (green) and Nitrogenase (rot). Big picture: Overlay, small picture: only nitrogenase (Immunostain)

Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294, 1534-1537

Inhibitor-Tests: Need of PSII-activity for nitrogen fixation in Trichodesmium

Influence of DCMU (10 µM), ascorbic acid (100 µM), and DTT (100 µM) wereInfluence of DCMU (10 µM), ascorbic acid (100 µM), and DTT (100 µM) were tested for cultures incubated under aerobic (white columns) and anaerobic (blue columns) conditions. Changes in nitrogenase activity as measured by acetylene reduction.

Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294, 1534-1537

Proof of Mehler-reaction during nitrogen fixation

Staining with DAB (Diaminobenzochinon) shows intracellular di t ib ti f H O b t i i ll lldistribution of H2O2 as brown stain in all cells

Mehler reaction: H O + 2O > H O +OMehler-reaction: H2O + 2O2 --> H2O2+O2

Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294, 1534-1537

) " b i ht"b i htl0

Diurnal cycle of activity: Distribution of F0 values

10

ntic

al c

ells

) "very bright"= bright type II

brighttype I

normal

low

F0

5bo

urin

g id

eNon-diazotrophic period

0

ps o

f nei

ghb

10

lls o

r gro

up

Diazotrophic period

5

obje

cts

(ce Diazotrophic period

0 10 20 30 40 50 600

Num

ber o

f o

0 10 20 30 40 50 60N

F0

Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135, 2120-33

Diurnal cycle of activity: correlation between bright cells, pigment content and nitrogenase activity

Nitrogenaseactivitye

activ

ityre

duct

ion)

)-1.m

in-1)

activityN

itrog

enas

em

l eth

ylen

e (m

l cul

ture

)

68

1012

N((

m .

% bright cells

ht c

ells

0246

% B

righ

M)

0 2

0.3

0.4

entra

tion

(µM

Chl

06 00 09 00 12 00 15 00 18 00 21 00 00 000.0

0.1

0.2

Chl

con

ce

06:00 09:00 12:00 15:00 18:00 21:00 00:00 control 5% oxygen 50% oxygen

Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135, 2120-33

PSII-activity Trichodesmium: reversibility of changes in fluorescence yieldy g y

Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135, 2120-33

Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant

Physiology 135, 2120-33

Hypothesis about the regulation of photosynthesis for nitrogen fixation in Trichodesmium

Licht initiates photosynthesis

Energy and reduction equivalent for CO2-Reduction of the PQ poolassimilation

Induction of a state 2 --> state 1-transition –cells have a high F0 (“bright cells type I”) due

to surplus phycobiliproteinsto surplus phycobiliproteins

Stimulation of pseudocyclic electron transport High respiration rates in early light period

Oxygen consumption exceeds oxygen production: Opportunity for nitrogen fixation

Carbohydrates are consumed

Respiration decreases, transition to the “normal F0 inactive” or “quenching” stateRespiration decreases, transition to the normal F0 inactive or quenching state

Intracellular oxygen rises

Nitrogenase becomes inhibited, no further nitrogen fixation until the following day, cells return to "normal F0 active" state

Purification of Trichodesmium phycobiliproteinsfor deconvoluting spectrally resolved in vivo fluorescence

kinetics and absorption spectrakinetics and absorption spectraPhycourobilin

isoforms Diazotrophic cell

eld

Phycourobilin isoforms

m)

basic fluorescence yield Fce

qua

ntum

yie

Phycobiliprotein purification + characterisation: Küpper H,

Andresen E, Wiegert S, Šimek M Leitenmaier B Šetlík I

isoforms (II)

axim

um)

o re

d m

axim

um

yield F0

ive

fluor

esce

nc

Method of deconvolution:

M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Phycoerythrinisoforms

alis

ed to

red

ma

(nor

mal

ised

to

eld

Rel

ati

Method of deconvolution:Küpper H, Seibert S, Aravind P

(2007) Analytical Chemistry 79, 7611-7627

Phycocyaninisoforms

scen

ce (n

orm

a

Fluo

resc

ence

PSII activity Fv

ce q

uant

um y

ie

Allophycocyanin

Fluo

res

ve fl

uore

scen

cR

elat

i

Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual

h bili t iphycobiliproteins

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual

ph cobiliproteinsphycobiliproteinsBasic dark-adapted fluorescence yield F0 Non-photochemical changes in fluorescence yield

quenching normal F0 active bright I =diazotrophic

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Sequence of uncoupling events in a „bright II cell“„ g

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167

Reversible coupling of individual

...as a basis for diazotrophic photosynthesis

phycobiliproteins...p y

Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim.

Biophys. Acta (Bioenergetics) 1787, 155-167

Iron limitation:Iron limitation: photosynthetic

componentspremain active...

Küpper H, Šetlík I, Seibert S, Prášil O, Šetlikova E, Strittmatter M, Levitan O, Lohscheider J, Adamska I, Berman-Frank I (2008) New Phytologist 179, 784-798

Iron limitation: rescue of photosynthetic components......by sacrificing nitrogenase

PsaCPsaC

new PE isoform

Küpper H, Šetlík I, Seibert S, Prášil O, Šetlikova E, Strittmatter M, Levitan O, Lohscheider J, Adamska I, Berman-Frank I (2008) New Phytologist 179, 784-798

Light limitation: acclimation by reversible phycobiliprotein coupling: basic fluorescence yield F0

relative frequency

Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2010) New Phytologist 185, 173-188

Light limitation: acclimation by reversible phycobiliprotein coupling (II): maximal PSII activity Fv

Andresen E, Adamska I, Šetlikova E, Lohscheider J, Šimek M, Küpper H, (2010) New Phytologist 185, 173-188

Conclusions

• For Trichodesmium, photosystem II activity is essential for providing energy for nitrogen fixation.

• Regulation of PSII activity for nitrogen fixation is achieved mainly by quickly reversible (un)coupling of individual phycobiliproteins.

• The nitrogen fixing activity state is characterised by a particularly large PSII-associated antenna, which is achieved mainly by coupling of additional units of phycourobilin (PUB) isoformsof phycourobilin (PUB) isoforms.

• Therefore, acclimation to light limitation (“low light stress”) involves enhanced th i i l f h bili hi h i th i l l d t PSIIsynthesis mainly of phycourobilin, which is then mainly coupled to PSII.

Synthesis and levels of other photosynthetic components decrease.

• Because of their vital importance, when adverse conditions require a choice, Trichodesmium in contrast to other cyanobacteria does not sacrifice its phycobilisomes, but rather its nitrogenase.p y , g

• Stress leads to expression of alternative phycobiliprotein isoforms

All slides of my lectures can be downloaded

from my workgroup homepage www uni-konstanz de Department of BiologyWorkgroups Küpper labwww.uni-konstanz.de Department of Biology Workgroups Küpper lab,

or directlyhttp://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html

and

on the ILIAS websiteon the ILIAS website