Tiago F. Jorge1, José C. Ramalho2,3, Ana I. Ribeiro-Barros1,2,3, Alisdair R. Fernie4 and Carla António1

1Plant Metabolomics Laboratory, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), Av. da República, 2780-157 Oeiras, Portugal2PlantStress&Biodiversity Lab, Linking Landscape, Environment, Agriculture and Food (LEAF), Dept. Recursos Naturais, Ambiente e Território (DRAT), Instituto Superior de Agronomia

(ISA), Universidade de Lisboa (ULisboa), Tapada da Ajuda, 1349-017 Lisboa, Portugal3GeoBioTec, Faculdade de Ciências e Tecnologia (FCT), Universidade Nova de Lisboa (UNL),

2829-516 Caparica, Portugal4Central Metabolism Group, Max Planck Institute of Molecular Plant Physiology,

D–14476 Potsdam-Golm, Germany

Casuarina glauca is a model actinorhizal plant characterized by its ability to establish

symbiosis with nitrogen-fixing Frankia bacteria. This plant species grows naturally in

coastal zones and is able to thrive under extreme salinity environments1,2. Due to such

strong resilience, C. glauca trees have been looked with a growing interest, mainly

because of the significant modifications in weather patterns over the past decades,

associated with climate changes.

C. glauca tolerance to high salinity is associated to biochemical and physiological

adjustments such as low tissue dehydration, osmotic adjustments, and high membrane

integrity3. To date, very little information is available in the literature about the C. glauca

metabolome. Mass spectrometry (MS)-based plant metabolomics has emerged as a

powerful tool to address biological questions related to plant environment and agriculture,

and therefore, the measurement of primary metabolites (e.g. sugars, amino and organic

acids) involved in the regulation of plant developmental processes has contributed to

better understand how plant metabolism readjusts in response to abiotic stresses4.

Important metabolites in plant responses to abiotic stress

GC-TOF-MS metabolite profiling

Fig.3- Neutral sugars of the raffinose family oligosaccharides (RFOs).

Fig.5- Quantification of raffinose in extracts of C.

glauca NOD+ plant tissues exposed to different

salinity stress levels (0, 200, 400 and 600 mM

NaCl). Values are mean ± SD of n=4-6 independent

PGC-LC-MSn measurements. FW, fresh weight. nd,

not detected.

Jorge et al (2017) Int J Mass Spectrom 413: 127–134

Fig.2- Heatmap showing metabolite responses in nodulated (NOD+) and non-nodulated

(KNO3+) C. glauca tissues. Relative values are normalized to the internal standard

(ribitol) and dry weight (DW) of the samples. Values presented as means ± SE of three

to five independent measurements. Dots indicate significant differences calculated using

the One-way ANOVA (p< 0.05) with respect to control. Grey-color squares represent not

detected (n.d) values. AA – amino acids, OA – organic acids, S – sugars; SA – sugar

alcohols.

A total of 39 and 37 primary metabolites (sugars and sugar alcohols, amino and organic acids) were

identified in NOD+ and KNO3+ C. glauca plants, respectively.

Fig.6- Quantification of raffinose in extracts of C.

glauca KNO3+ plant tissues exposed to different

salinity stress levels (0, 200, 400 and 600 mM

NaCl). Values are mean ± SD of n=4-6 independent

PGC-LC-MSn measurements. FW, fresh weight. nd,

not detected.

Jorge et al (2017) Manuscript under revision

INTRODUCTION

C. glauca plants

Nodulated (NOD+) Non-nodulated (KNO3+)

Root-nodules

Branchlets

N2 (Frankia Thr) Mineral nitrogen KNO3+

Roots

Branchlets

0, 200, 400 and 600 mM NaCl

Plant tissues

N-source

Stress

conditions

EX

PE

RIM

EN

TA

L D

ES

IGN

RESULTS

MS-based metabolomic analyses

LC-MS/MS target analysis of RFOs

Raffinose Stachyose Verbascose

RFOs Positive ion mode ESI-QIT-MSn structural characterization

Quantification of RFOs in C. glauca plant tissues

Fig.1- Principal component analysis (PCA) score plots of metabolic profiles

in C. glauca tissues: (a) PCA score plot for root-nodules of NOD+ and roots

of KNO3+ and (b) PCA score plot for branchlets of NOD+ and branchlets of

KNO3+.

Fig.4- Positive ion CID spectra of raffinose obtained by PGC-ESI-QIT-MSn showing nomenclature of

Domon and Costello5: (a) full MS spectrum of raffinose ([M+Na]+ m/z 527); (b) CID MS2 spectrum of

raffinose (precursor ion [M+Na]+ m/z 527); (c) CID MS3 spectrum of raffinose (precursor ion [M+Na]+ m/z

365). The italics, bold and bold-italics correspond to the Ci, Bi and Ai ions according to the nomenclature

of Domon and Costello5.

- Our MS-based metabolomics approach provides new knowledge regarding the primary metabolome of

nodulated (NOD+) and non-nodulated (KNO3+) C. glauca plants.

- Our results agree with those previously obtained from morpho-physiological analysis6,7.

- The main differences observed in the metabolite pool between NOD+ and KNO3+ plants not only rely on

the impact of the salt stress itself, but also on the symbiotic activity damage of the NOD+ plants at early

salt stress exposure.

CONCLUSIONS

REFERENCES

A second independent biological experiment is currently ongoing to assess, at the physiological and

metabolite levels the performance of non-nodulated C. glauca plants under a combined salt and heat

stress.

ONGOING WORK1. Zhong, C, Mansour, S, Nambiar-Veetil, M, Boguz, D & Franche, C (2013) J. Biosci. 38, 815–823

2. Pawlowski, K & Demchenko, KN (2012) Protoplasma 249, 967–979

3. Ribeiro-Barros, AI et al. (2016) Symbiosis 70, 111–116

4. Jorge, TF et al. (2016) Mass Spectrom. Rev. 35, 620–649

5. B Domon, CE Costello (1988) Glycoconjugate J. 5: 397–409

6. Batista-Santos, P et al. (2015) Plant Physiol Biochem 96, 97–109

7. Duro, N et al. (2016) Plant Soil 398, 327–337

This work was supported by the FCT Investigator Programme (IF/00376/2012/CP0165/CT0003) from Fundação

para a Ciência e a Tecnologia and the ITQB NOVA R&D unit GreenIT (UID/Multi/04551/2013). T.F.J. gratefully

acknowledges FCT (PD/BD/113475/2015) and the ITQB NOVA International PhD Programme ‘Plants for Life’

(PD/00035/2013) for the PhD fellowship. A.I.R.-B. acknowledges FCT under the scope of the project PTDC/AGR-

FOR/4218/2012. C.A. gratefully acknowledges the Portuguese Mass Spectrometry Network (Rede Nacional de

Espectrometria de Massa, RNEM) for support.

ACKNOWLEDGMENTS