Regulation of Photosynthesis for Nitrogen Fixation

Regulation of Photosynthesis for Nitrogen Fixation
Hendrik Küpper, visit to Aberdeen, February 2014
Nitrogen cycle: intermediate steps
NO3Nitrite oxidase
Nitrate reductase
NO2-
NO
N2O
Anammox
H2NOH
N2
NH3
Total equation of biological nitrogen fixation
N2 + 8H+ + 16 MgATP + 8e-
2NH3 + H2 + 16 MgADP + 16 Pi
Organisms involved in nitrogen fixation
Anabaena
Azolla
Trichodesmium
Gloeothece
Rhizobien
Azotobacter
Mechanism of biological nitrogen fixation:
nitrogenase of cyanobacteria
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 cyanobacteria
y
Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_157-164
Unicellular cyanobacteria
Regulation of photosynthesis for nitrogen fixation in
unicellular cyanobacteria
y
((II))
glycogen storage begins
max. photosynthetic capacity
light period:
carbon + energy storage
down regulation of
photosynthesis
maximum glycogen storage
up regulation of
y
photosynthesis
nitrogen fixation begins
dark period: nitrogen fixation
maximum nitrogen fixation
maximum 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
parameters
M i l flfluorescence quantum
Maximal
t
yield
i ld (Fm)
25 µm
13 h
36 h
55 h
112 h
Photosystem II activity (Fv/Fm)
Ferimazova N, Felcmanová K, Šetlíková E, Küpper H, Maldener I, Hauska G, Šedivá B, Prášil O (2013) PhotRes 116, 79-91
Heterocyst differentiation:
changes in parameters of chlorophyll fluorescence kinetics
Ferimazova N, Felcmanová K, Šetlíková E, Küpper H, Maldener I, Hauska G, Šedivá B, Prášil O (2013) PhotRes 116, 79-91
Trichodesmium:
anoxygenic photosynthesis energizing nitrogen fixation
i the
in
th same cells
ll during
d i the
th photoperiod
h t
i d
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 photosynthesis
photosynthesis.
• How nitrogenase is protected from damage by
photosynthetically produced O2 has remained an enigma.
Surface blooms in the
Arafura Sea
Colonies – tuft and puff
formation
Trichodesmium
35
1.0
30
25
0.9
20
0.8
15
colonies: “Tuft” and
“Puff”
GP (µM O2 µg chl a-1 h-1)
0.7
10
0.6
5
0.5
0
60
-2
50
-4
40
-6
-8
30
-10
10
20
-12
10
-14
0
-16
-10
-18
-20
-20
8
10
12
14
16
18
20
Nitrogen
n fixation
(n
nmol C2H2 µ
µg chl a -1 h-1)
11
1.1
Dark rrespiration
(µM O2 µ
µg chl a-1 h-11)
TrichodesmiumT
i h d
i
bloom
Relativee Fv/Fm
Aktivitätszyklus von Trichodesmium
22
Local tim e ((h))
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) 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 off H2O2 as brown
distribution
b
stain
t i iin allll cells
ll
Mehler reaction: H2O + 2O2 -->
Mehler-reaction:
> H2O2+O2
Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294, 1534-1537
low F0
Number of o
N
objects (cells or group
ps of neighb
bouring identical cells))
Diurnal cycle of activity: Distribution of F0 values
10
normall
bright
b
i ht
type I
""very bright"
b i ht"
= bright type II
Non-diazotrophic period
5
0
10
Diazotrophic period
5
0
0
10
20
30
40
50
60
F0
Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135, 2120-33
Nitrogenase
N
e activity
Chl conce
entration (µM
M) % Brigh
ht cells ((m
ml ethylene reduction)
-1
-1
.(ml culture)) .min )
Diurnal cycle of activity: correlation between bright cells,
pigment content and nitrogenase activity
12
10
8
6
4
2
0
0.4
Nitrogenase
activity
% bright cells
Chl
0.3
02
0.2
0.1
0.0
06 00
06:00
09 00
09:00
12:00
12
00
control
15:00
15
00
18 00
18:00
5% oxygen
21:00
21
00
00 00
00:00
50% oxygen
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
Reduction of the PQ pool
Energy and reduction equivalent for CO2assimilation
Induction of a state 2 --> state 1-transition –
cells have a high F0 (“bright cells type I”) due
to 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
normal F0 inactive
inactive” or “quenching”
quenching state
Intracellular oxygen rises
Nitrogenase becomes inhibited, no further nitrogen fixation until the following day, cells return to
"normal F0 active" state
Deconvolution of spectrally resolved in vivo fluorescence
kinetics shows reversible coupling of individual
ph cobiliproteins
phycobiliproteins
Basic 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
g II cell“
Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787, 155-167
Reversible coupling
of individual
phycobiliproteins...
...as a basis for diazotrophic
photosynthesis
p
y
Küpper H, Andresen E, Wiegert S, Šimek M,
Leitenmaier B, Šetlík I (2009) Biochim.
Biophys. Acta (Bioenergetics) 1787, 155-167
Iron limitation: rescue of photosynthetic components...
...by sacrificing nitrogenase
PsaC
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
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 PSIIassociated antenna, which is achieved mainly by coupling of additional units
of phycourobilin (PUB) isoforms
isoforms.
• Therefore, acclimation to light limitation (“low light stress”) involves enhanced
synthesis
th i mainly
i l off phycourobilin,
h
bili which
hi h iis th
then mainly
i l coupled
l d tto PSII
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,
p
y
, but rather its nitrogenase.
g
• Stress leads to expression of alternative phycobiliprotein isoforms
Current and former lab members
involved in this project
Elisa Andresen, Barbara Leitenmaier, Qiyan Wang-Müller, Sven Seibert,
Verena Rösch, Christian Lang
Collaborators
Naila Ferimazova, Ivan Šetlík, Eva
Iwona Adamska, Jens
Š lík
Šetlíkova,
Ondrej
O d j Prašil:
P šil Ph
Photosynthesis
h i
Lohscheider, Martina
Research Centre, Institute of
Strittmatter: Universität
Microbiology, Třeboň, Czech Republic
y
Konstanz,, Germany
Ilana Berman-Frank: Bar
Ilan University, Israel
Pernilla Lundgren,
Lundgren Birgitta
Yi Bu Chen,
Yi-Bu
Chen Zbigniew Kolber,
Kolber Paul
Bergman: Stockholm University
Falkowski:
Sweden
Rutgers University, U.S.A
Main sources of external funding
Deutsche Forschungsgemeinschaft
NATO “Science for Peace” programme
Ministry of Education of the Czech Republic
All slides of my lectures can be downloaded
from my workgroup homepage
www uni-konstanz de  Department of Biology  Workgroups  Küpper lab
www.uni-konstanz.de
lab,
or directly
http://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html
and
on the ILIAS website

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