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

Morphological observation

*

* Days of observation after exposure to SMF for 10 days

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) mariaelena.villani@enea.it

Thanks

for your attention