METABOLIC CHANGES AND PHYSIOLOGICAL RESPONSES
INDUCED BY SPACE CONDITIONS
IN CHLAMYDOMONAS REINHARDTII D1 MUTANTS
National Research Council
(CNR) Rome
Soyuz-U, Foton M2, 3
Space Mission 2005-2007
Endeavour, STS-134
Space Mission 2011, 2016
Kennedy Space Center, NASA
Florida
Baikonur Cosmodrome,
Kazakhstan
MARIA TERESA GIARDI
Plant Biotechnology
Technological Applications of Photosynthesis and
life support systems in Space
Edible Biomass
Vitamins, antioxidants
Oxygen Production
BIOFUEL
Photosynthetic BIOMASS and Metabolites with Recognized
Radical Scavenging Properties
MT Giardi, S Silletti, L Lastella. “Process for production of algal biomass". International Patent 2012, BWO15039, PCT/IB2012/057336
Lavecchia T, Rea G, Antonacci A, Giardi MT. Healthy and Adverse Effects of Plant-Derived Functional. Critical Review in Food Science and Nutrition. 2013. DOI:10.1080/10408398.2010.520829.
Rea G, Antonacci A, Lambreva M, Pastorelli S, Tibuzzi, A, Ferrari S, Fischer D, Johanningmeier et al. Giardi MT. (2011) Integrated plant biotechnologies applied to safer and healthier food production: the Nutra-Snack manufacturing chain. Trends in Food Science & Technology 22 , 353-366.
Photosynthetic organisms are the basis of any life support system
•they produce O2 for astronauts, remove CO2 from the atmosphere and can be used as food supplement.
However
•they are affected on long-term missions by cosmic radiation which generates dangerous reactive oxygen species (ROS) in the cell. Also microgravity affects their performance. In Space it is not possible to distinguish the two effects.
As a consequence
•they have to be adapted to space conditions in order to be more robust and reliable
Cano J, Giannini D, Pezzotti G, Rea G, Giardi MT. (2011) Space impact and technological transfer of a biosensor facility to Earth application. Recent Patents on Space Technology. Vol.1, 5-10
a unicellular green alga having:
short life cycle
easy cultivation
huge mutant collections
Chlamydomonas in Space
easily transformable
Microalgae are not adapted to the conditions in
spacecrafts and planetary environments
Natural evolution takes millions of years…
Side-directed mutagenesis takes a few weeks
OUR APPROACH Directed Evolution
Exposure to Space ionizing radiation can damage
• DNA• Proteins• Pigments• Lipids
In photosynthesis, an important targetfor radiation damage is photosystem II
Radiation generates reactive oxygen species(ROS), thus destroying the D1 subunit of PSII
The steps of in vitro Accelerated Evolution Approach
Selection for photo-autotrophic growth
2. Selection by exposure to ionizing radiation
neutrons, 14 MeV (ENEA)
doses: 35&75 mGy
protons, 27 MeV (INFN)
doses: 0.5&5 Gy
1. Random mutagenesis
Error-Prone PCR on psbA gene
psbA
xx
x
x
x
Transformation by particle gun
psbA
Primary pool of mutants
Cocktail ofD1-random mutants
3. Identification of the D1 mutations
in survived colonies
psbA gene sequencing analyses
Several of these strains hosted
aminoacidic substitutions near to
TyrZ, OEC and the QB binding
site
Among the selected mutants
Several mutants were identified
and characterised
Site-directed mutagenesis
Ionizing radiation tolerant strains:
from random towards site-directed mutants
Giardi MT, Rea G, Lambreva MD, Antonacci A, Pastorelli S,
Bertalan I, et al. (2013) Mutations of Photosystem II D1 Protein
That Empower Efficient Phenotypes of Chlamydomonas
reinhardtii under Extreme Environment in Space. PLoS ONE
8(5): e64352. doi:10.1371/journal.pone.0064352
Mission duration:
Sept. 14 (11:00 UT) to Sept. 26 (07:58 UT), 2007
Orbital period: 90 minutes
Completed orbits: 189
Maximum altitude: 302 km
Minimum altitude: 263 km
Inclination: 63°
FOTON- M1,2,3 SPACE MISSIONS
Mutants tested in D1 protein of Photosystem II
Strains Amino acid substitution or insertion in D1
IL psbA → gene without introns, considered as a
reference strain
I163N Ile163 → Asn near to Tyr 161, the primary
electron donor of P680.
S264K Ser264 → Lys in binding niche for QB
resistant against different class of herbicides
A250L Ala250 → Leu in binding niche for QB
A251C Ala251 → Cys in binding niche for QB
Resistant against different class of herbicides
Is it possible to consider the D1
protein a molecular target to
improve the space ionizing
radiation tolerance?
Rea G, Esposito D, Damasso M, Serafini A, Margonelli A, MT Giardi (2008) Ionizing radiation impacts photochemical quantum yield and oxygen evolution activity of Photosystem II in photosynthetic microorganisms. Int J Radiat Biol 84: 867–877
Kindly provided by Prof. Niyogi
Strains Mutations in carotenoids biosynthetic
pathway
cc125 Wild type (www.chlamy.org)
npq2 Impaired in zeaxanthin epoxidase
anable to convert zeaxanthin in antheraxantin
lor1 Impaired in lycopene e-cyclase
unable to make α-carotene and lutein
npq2 lor1 Impaired in zeaxanthin epoxidase and lycopene
e-cyclase, accumulation of zeaxanthin and
-carotene
Other tested mutants in the carotenoid pattern of Chlamydomonas
Prolycopene
Phytoene
-carotene
Lycopene
psy
pds
-carotene
-carotene Lutein
lyc-
Zeaxanthin
Antheraxanthin
Violaxanthin
lyc-b chy-b
zep
zep vdr
vdr
chy-b
Geranylgeranyl-diphospate
What’s the role of antioxidants on
protection against space environment
The mutants I163N, A251C and npq2lor1 were
the most stable strains in flight and after landing
MUTANTS WITH SIMILAR STABILITY
IN FLIGHT IL, I163N, S264K, A250L, A251C, cc125, npq2lor1
AFTER LANDING IL, I163N, A251C, npq2lor1
Photosystem II performance
Liulin-Photo
Photo-II
Survival cell unit,
white LEDs
Measuring cell unit,
red and white LEDs
Multicell
container
chambers with
algae on agar
7 h light/17 h dark
fluorescence measurement
every hour
thermo-sensors
data storage for 1 month
FOTON SPACE MISSIONS 2005-2007
Rea G, Lambreva M, Polticelli F, Bertalan I, Antonacci A, Pastorelli S, Johanningmeier, Giardi (2011)
Directed Evolution and In Silico Analysis of Reaction Centre Proteins Reveal Molecular Signatures of
Photosynthesis Adaptation to Radiation Pressure. PLoS ONE 6(1): e16216.
doi:10.1371/journal.pone.0016216
PHOTO II hourly measurements
Giardi, Rea, Lambreva, Antonacci, Bertalan, Johanningmeier, Mattoo.Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of Chlamydomonas reinhardtii under Extreme Environment in Space. 2013. PLoS ONE 8(5): e64352. doi:10.1371/journal.pone.0064352
Daily trend of Fv/Fm in flight
cc125 Strain a wild-type with reduced activity in flight and
after flight
Fv/Fm
In Flight After Landing
Flight stable (0.65-0.68) decrease steeply in comparison to the
control
Control stable (0.71-0.78) stable
Daily trend of Fv/Fm in flight
A mutant npq2lor1 Strain maintaining high activity
Fv/Fm
In Flight After Landing
Flight stable (0.63-0.67) stable (0.57-0.61)
Control stable (0.63-0.67) stable (0.60-0.64)
geranylgeranyl-diphospate (GGPP)
O P O P OH
OH OH
ISOPENTENYL DIPHOSPHATE
(IPP)
GGPS
phytoene
psy
carotene
pds
lycopene
zds
carotene carotene
lyc blyc e
lutein
chy b chy b
zeaxanthin
antheraxanthin
violaxanthin
vdr
vdr
zep
zep
p-hydroxyphenilpyruvate
2-methyl-6-phytyl-1,4-benzoquinone
hst 1
homogentisate
Plastoquinone-9
mpbq-mt
3
psbA & psbD
Biosynthetic Pathways analysed by real time
sqRT-PCR Analyses
Rea, Giardi, Antonacci, Lambreva et al unpublished
Strains lycb lyce chyb pds vdr zep
IL -4,10 -1,09 -6,17 -2,28 1,16 1,42
I163N -1,67 -1,05 2,17 -4,45 -1,49 -1,77
A251C -2,27 1,27 -1,98 -3,27 -5,15 -3,19
A250L -1,33 -2,17 -7,21 -6,68 -2,24 -2,57
cc125 8,63 4,03 2,32 -1,60 -1,11 1,57
npq2lor1 1,14 -1,59 4,64 -4,33 -11,24 -1,06
Space environment strongly affects carotenoid, plastoquinone and reaction centre D1 protein biosynthetic pathways
sqRT-PCR Analyses of the Carotenoid genes
Rea, Giardi, Antonacci, Lambreva et al unpublished
STS-134 SPACE MISSION
Launch:
8:56 a.m. EDT - May 16, 2011
Landing:
2:34 a.m. EDT - June 1, 2011
Orbiter:
Endeavour
Mission Number:
STS-134
(134th space shuttle flight)
Mission Duration:
16 days
Landing Site:
KSC
Inclination/Altitude:
51.6 degrees/122 nautical miles
Selected Strains for the
flight
I163N I163T P162S
STS-134 Space Mission:
BIOKIS-PHOTOEVOLUTION project
The mutants I163N, I163T and P162S, with the parental
strain IL, took part in the BIOKIS project enclosed in the
PHOTO I hardware, as
PHOTOEVOLUTION experiment
Daily neutron dose
Ground-Average 1 mSv/day
Flight-Average 17.2 mSv/day
Vukich, Ganga , Cavalieri, Rizzetto, Rivero, Pollastri, Mugnai, Mancuso, Pastorelli, Lambreva, Antonacci, Margonelli, Bertalan, Johanningmeier, Giardi, Rea, Pugliese, Quarto, Roca; Zanini, Borla,Rebecchi, Altiero; Guidetti, Cesari, Marchioro, Bertolani, Pace, De Sio, Casarosa, Tozzetti, Branciamore, Gallori, Scarigella, Bruzzi, Bucciolini, Talamonti, Donati, Zolesi. BIOKIS: a model payload for multidisciplinary experiments in microgravity. Microgravity Science and Technology, June 2012 DOI 10.1007/s12217-012-9309-6
• psbA gene expression levels were increased
• psbD gene expression was down regulated
QBQA
TyrZ
P680
psbD psbA
PSII core complex
D1D2
protein heterodimer
Transcript profile of psbA and psbD gene after
the flight
Rea, Giardi, Antonacci, Lambreva et al unpublished
PHOTOEVOLUTION: SHORT TERM-FLIGHT
The control IL is strongly affected by the flight and died while the selected mutants showed high performance
Conclusions – Foton and ISS Missions
Radiation tolerance in I163N, A251C and npq2lor1 was probable dueto antioxidant pigments accumulation (b-caroten, zeaxanthin andlutein)
Down regulation of gene involved in carotenoids biosynthetic pathway could indicate negative feedback regulation
Plastoquinone biosynthetic pathway was strongly down regulated, possible product of negative feedback regulation
The enhanced mutants capability to survive to the cosmic adverse conditions has been related to the particular localization of the aminoacid substitution in the D1 photosynthetic structure
psbA mRNA accumulation probably indicates the high protein turn-over of D1 protein due to the harsh radiation environment
EXPECTED LONG-TERM ISS EXPERIMENTSD1 random mutants tolerant to neutron/proton
bombardments
Under neutron mainly aliphatic and aromatic residues
Under proton only aliphatic and aromatic residues
Among the 2000 produced strains, 19 overcame the radiation induced stress
Mutants Amino acid
substitutions
Amino acid properties
hydropathy indexa/reactivity class/side chain polarity
Localization of the mutation
in the protein
wild type → mutated wild type → mutated
P162S proline serine -1.6 (III) nonpolar -0.8 (0) polar near to Tyr161
I163T isoleucine threonine 4.5 (IV) nonpolar -0.7 (0) polar near to Tyr161
M172L methionine leucine 1.9 (V) nonpolar 3.8 (IV) nonpolar near to OEC
G207S glycine serine -0.4 (I) nonpolar -0.8 (0) polar in the helix IV of D1
L200I leucine isoleucine 3.8 (IV) nonpolar 4.5 (IV) nonpolar in the helix IV of D1
I281T isoleucine threonine 4.5 (IV) nonpolar -0.7 (0) polar in the helix V of D1
Most of the amino acid substitutions consisted of replacement of non-polar with polar residues that are less prone to oxidative damage.
D1 site-directed mutants of the randomtolerant strains to radiation
The mutants show similar growth rate but accumulate much less chlorophyll than the
reference strain
Photoheterotrophic growth
Maximum quantum yield (Fv/Fm) and electron transport efficiency
(1-Vj) of the mutants are lower compared to the reference strain IL
PSII Photosynthetic efficiency
In high photon fluency conditions
(150 mmol m-2 s-1) mutants maintain
a higher stability of photosynthesis
efficiency compared to the reference
strain strain, IL
In high photon fluency conditions
mutants have a higher oxygen evolution
capacity compared to the reference
strain, IL
PSII photochemistry & Light-dependent oxygen evolution
capacity at high photon fluency rate of the mutants
We found that D1 protein of photosystem II displays asignificant lower content of non-polar, oxidative damagesensitive, residues along the evolution scale suggesting ahigher radiation pressure today than in archea
Results
BIOINFORMATIC ANALYSIS:In silico analysis of the L/D1 amino acid sequences of
Bacteria, Cyanobacteria and Eukarya
In conclusionProposed Experiments in a long-term flight to ISS
expected in 2016
Testing the mutants above, selected to be resistant to neutrons and protons
Construction of algal mutants which overexpress enzymes and/or peptides which reduce the amount of ROS caused by cosmic radiation
Overexpression of superoxide dismutase (SOD), glutathione peroxidase (GPX), ascorbate peroxidase (APS) or methionine sulfoxide reductase (MSR)
Overexpression of antioxidative peptides
Several mutants have been generated already and are available for the project
The team
For 2016 ISS flight
Maria Teresa Giardi
Mehmet Turemis
Gianni Pezzotti
Prof. Udo Johanningmeier
Dr Ivo Bertalan
Thanks
Partners from the Industry
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