Effects of high-intensity static magnetic fields
on an in vitro plant system for the production of
biopharmaceuticals in aerospace environment.
Maria Elena VILLANI ENEA, Casaccia Research Center
Biotechnologies and Agro-Industry Division
Via Anguillarese 301 - 00123 Rome (Italy)
7th International AgroSpace Workshop
Mars – A Long Way to Go
Sperlonga (LT)
May, 26th – 27th 201622
Effects of high-intensity static magnetic fields
on an in vitro plant system for the production of
biopharmaceuticals in aerospace environment.
BIOxTREME Project
Plant BIOfactories for the formulation of bioactive molecules with microbicidal, immunostimulating and antioxidant activity
for life in exTREME conditions
2/37
Space exploration
Long term missions
Availability of resources for human survival
(food and medicine)
Problems of supply from Earth:
• weight and size of the load
• time expiration
• no fresh food
Plant as a source of
• fresh food
• bioactive molecules which may
counteract the deleterious effects of
permanence in confined environments
exposed to physical stress
The space … in the laboratory
Proton beams ENEA-Physical
Technologies for Safety
and Health Division
Static magnetic
fields (SMF) ENEA-Health Protection
Technology Division
Gamma rays ENEA‐National
Institute of Ionizing
Radiation Metrology
(INMRI)
Microgravity ENEA-Biotechnologies
and Agro-Industry
Division
Magnetic fields in space
European Space Radiation Superconducting Shield (SR2S)
CERN project developing a superconducting magnet to shield spacecraft
and protect astronauts from cosmic radiation (high-energy particles)
during deep-space missions.
Magnetic fields in space
European Space Radiation Superconducting Shield (SR2S)
CERN project developing a superconducting magnet to shield spacecraft
and protect astronauts from cosmic radiation (high-energy particles)
during deep-space missions.
SMFs in the order of some hundreds mT, possibly exceeding the exposure
limits for general public established by international guidelines (*), could
be experienced within a spacecraft.
(*) International Commission on Non-Ionizing Radiation Protection (ICNIRP), “Guidelines on limits of exposure to static magnetic fields”, Health Phys.
(2009) 96(4): 504-514.
Effects of static magnetic fields (SMF) on plants
Natural static magnetic field of the Earth: 50 μT
The highest non-occupational exposure in patients undergoing a magnetic
resonance: from 0.15 to 3 T (for about 1 h)
Occupational exposure limits: 2 T (8 T for limbs)
Effects of static magnetic fields (SMF) on plants
Main documented effects at SMF from 50 mT to
250 mT concern growth, development, redox
status:
• Stimulation of seed germination and early
growth
• Induction of α-amylase, dehydrogenase and
protease in seeds
• Induction of root proliferation and curvature
During evolution plants have experienced
variations and inversions of the geomagnetic
field, revealing adaptive ability
Review: Maffei ME. Front Plant Sci. 2014; 5: 445
Plant system chosen for BIOxTREME
Tomato hairy root culture
genetic engineering mediated by soil bacteriun
Agrobacterium rhizogenes, able to induce neoplastic
proliferation and the production of protein of interest
(antifungal and anticancer antibodies)
Hairy roots expressing the transcription factor
AN4 (c-Myb) inducing anthocyanin synthesis
University of Amsterdam, Prof. F. Quattrocchio
ANTHOCYNINS
? AN4
Anthocyanins protect plants against
oxidative stress (i.e. ionizing radiation)
Hong MJ et al. Int J Radiat Biol. 2014; 90:1218-28
How do tomato hairy roots react in a SMF ?
500 mT 250 mT blank
Poster: ‘Exposure devices for investigating effects of high intensity static magnetic fields on an in vitro plant
system for the production of biopharmaceuticals in aerospace environment. Lopresto et al.
acute exposure 1 day
chronic exposure 10 days
× 3 replicates
2D–DIGE proteomics
Proteomics analysis
Differential proteomic approach was adopted to analyze the response of the plant tissue comparing
the expression profiles at different intensities of SMF and at increasing exposure times.
DIGE (Difference
In Gel
Electrophoresis)
An average of 900 protein spots were detected in each gel and the spot analyzed
with Decyder software did not change in a significant way from sham to 250 and
500 mT for both 1 and 10 days of exposition.
Differential In Gel Electrophoresis (DIGE) analysis
Gel 1 Gel 2 Gel 3
Gel 4 Gel 5 Gel 6
Gel 7 Gel 8 Gel 9
2D-DIGE statistical data elaboration
Number of spots per gel ≈ 900
Statistical parameters:
1-ANOVA p ≤ 0.05, fold change ≥ 1.5
When FDR correction was applied
no differential spots were evidenced
Without FDR filter 37 differential spots
were obtained
17 protein spots were identified by
mass spectrometry
1555
1035 1056 1057
1190 1324
1718
682 747 743
745
677
2182
4495 4512
1304
2022
pI 4.0 7.0
kD
a
12
0
14
N. Spot Average Ratio 1-ANOVA
500 mT 10 days vs sham 10 days 563 2.08 0.014 564 2.81 0.018 682 2.90 0.003 745 -1.84 0.035 874 2.51 0.045
1088 2.01 0.026 2022 1.65 0.047
250 mT 10 days vs sham 10 days 314 2.01 0.039 563 1.76 0.014 564 2.24 0.018 639 -1.71 0.014 677 2.40 0.023 743 3.44 0.022 745 -1.52 0.035 747 -2.37 0.047 874 2.82 0.045
1056 -2.89 0.033 1057 -1.67 0.033 1088 2.15 0.026 1170 1.68 0.016 2022 1.80 0.047
500 mT 1 day vs sham 1 day 563 -1.79 0.014
3361 -1.66 0.012 4377 -1.73 0.027
250 mT 1 day vs sham 1 day 563 -1.93 0.014 639 1.82 0.014 564 -1.84 0.018
3361 1.51 0.012 1056 1.67 0.033
N. Spot Average Ratio 1-ANOVA
all 10 days vs all 1 day 4495 -3.83 0.013 682 -3.03 0.003
1007 -2.49 0.017 630 -2.46 0.015
1057 -3.10 0.033 1423 -2.12 0.034 1428 -1.99 0.028 747 -3.18 0.047
1056 -3.51 0.033 1088 -2.42 0.026 1508 -1.64 0.048 743 -4.02 0.022
2182 -1.85 0.049 822 -1.75 0.014
1097 -2.03 0.048 1410 -1.86 0.043 745 -3.1 0.035
4512 -3.34 0.021 1718 -1.98 0.040
Differentially expressed spots
1190 -1.94 0.039 1324 -2.45 0.049 1555 -2.6 0.046 639 -2.47 0.014
1574 -3.25 0.033 4377 -2.18 0.027 2022 -1.73 0.047 1035 -1.94 0.037 4562 -1.92 0.033 1170 -1.59 0.016 730 -2.05 0.048 314 2.01 0.039
1304 -1.83 0.037 677 -1.91 0.023 874 -2.16 0.045
3361 -2.33 0.012
vs
7 spots
14 spots
3 spots
5 spots
35 spots
vs 10 1
Protein identification by MS
Sugar metabolism 677, sucrose synthase
Amino acid metabolism 682, 743,745, 747 homocysteine
methyltransferase
Energy 1035 ATP-citrate lyase, 1056 phosphoglycerate mutase
1057 phosphoglucomutase, 1190 pyruvate kinase
1324Enolase, 1555,1718 Isocitrate dehydrogenase
Disease/defence 2182 peroxidase
4495, 4512 MLP-like protein
Protein synthesis 1304, seryl-tRNA synthetase
2022 60S acidic ribosomal protein
Proteomic multivariate analysis
Not evident separation
of the groups
Principal Component Analysis
Hierarchical Clustering Analysis
Proteins are mainly
grouped for the exposure
time and not for the
intensity of SMF exposure
CONCLUSIONS and PERSPECTIVES
• Exposure to static electromagnetic fields, of comparable intensity
to those that would be created in magnetically shielded space
habitats, seems not to significantly alter the physiology of the plant
system examined. The observed variations may be considered
rather an expression tendency than a change with functional effects.
• The final aim of BIOxTREME project is to
demonstrate that this plant ideotype could be
useful as a bioreactor for the production of
ready-to-use bioactive molecules (i.e. anti-
mycotic antibodies) during long term space
missions.
• The exposure to other stressors characterizing extraterrestrial
environments (i.e. ionizing radiations) will assess if the expression
trend observed for SMF might be synergistically reinforced, leading
to a significant alteration of the expression profiles.
Acknowledgements
Working group for SMF:
Eugenio BENVENUTO
Angiola DESIDERIO
Silvia MASSA
Cristina CAPODICASA
Vanni LOPRESTO
Rosanna PINTO
Italian Space Agency
National Research Council - Naple
Proteomics & Mass Spectrometry Laboratory:
Andrea SCALONI
Anna Maria SALZANO
Maria Elena Villani
ENEA Casaccia Biotechnologies and Agro-Industry Division (SSPT-BIOAG)
Via Anguillarese 301, 00123 Rome (Italy) [email protected]
Thanks
for your attention