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Mapping of P and K nutrients using X-ray fluorescence: first insights on P exchanges in ectomycorrhizal roots
Claude Plassard, Camille Rivard, Laurie Amenc, Carlos Trives Segura, Benedickt Lassalle
To cite this version:
Claude Plassard, Camille Rivard, Laurie Amenc, Carlos Trives Segura, Benedickt Lassalle. Mapping of P and K nutrients using X-ray fluorescence: first insights on P exchanges in ectomycorrhizal roots. 3. Adam Kondorosi Symposium, Apr 2017, Gif-sur-yvette, France. 2017, Frontiers in Beneficial Plant-Microbe Interactions. �hal-01608334�
0 200 400 600 800 1000 1200 1400 0 20 40 60 80 100 120 140 P number of counts (x10 000) Cytosol (HN) Vacuole Vacuole Cytosol (FS) Cc Cx Cx Bkg Bkg P number of counts (x10 000) 0 100 200 300 400 500 600 0 30 60 90 120 150 Pixels nucleus Cytosol Vacuole Vacuole Cytosol Cc Cx Cx Bkg Bkg fit
Samples are moved relative to the focused beam to scan the area of interest and an XRF spectrum is recorded in each pixel of the map.
Elemental
co-localization map
Elemental mapping
max
min
Mapping of P and K nutrients using X-ray fluorescence:
first insights on P exchanges in ectomycorrhizal roots
C. Plassard
1, C. Rivard
2,3, L. Amenc
1, C. Trives
1, B. Lassalle
3claude.plassard@inra.fr camille.rivard@synchrotron-soleil.fr 1. Eco&Sols, INRA-IRD-CIRAD-SupAgro, 34060 Montpellier
2. CEPIA, INRA, 44300 Nantes
3. Synchrotron SOLEIL, 91190 Gif-sur-Yvette
It is well known that ectomycorrhizal (ECM) fungi improve the growth of their hosts by providing growth-limiting nutrients and particularly
inorganic phosphate (Pi). However, the molecular players driving the Pi allocation from the fungus to the host are largely unknown thus far.
The objective of this study was to understand the role of a candidate fungal P transporter gene for P efflux (Hc-PT2) in the ECM association
between Pinus pinaster and the basidiomycete Hebeloma cylindrosporum. To fill this objective, we compared the P distribution in short roots
of non mycorrhizal (NM) plants with those found in ECM roots associated with a fungal strain previously transformed with an
over-expression vector, either containing no gene (Ctrl) or our candidate gene (OE-PT2).
Context and Objective of the study
X-ray Eexc Energy continuum continuum M L 2. Electronic transition K 1. Ionization Ka Kb Energy specificity of the reemitted X-ray M L K Photoelectron
X-ray spectroscopy fluorescence principle
Elemental composition determination
X-Ray Eexc
Energy dispersive detector
XRF intensity Fe Ca Cl P Qualitative and quantitative information Heterogeneous sample XRF spectrum
Elemental mapping using X-ray fluorescence (XRF) on LUCIA beamline
Plants growth and samples preparation
Elemental distribution of P, K, Cl and Mg in pine tree roots
XRF maps were recorded using a 3 x 3 µm² pixel size at 3.7 keV to collect signal of K, Cl, S, P, Si, Al and Mg.
To compare the levels of P and K along the different growth conditions, P and K XRF values of the pixels were extracted along six profiles using ImageJ.
Several locations can be distinguished as a function of the pixel value: background (Bkg), cortex (Cx), central cylinder (Cc) and cortical cytosol and vacuoles in non mycorrhizal (NM) root, accordingly to the pixel values.
P
PyMCA software (Solé et al. 2007)
P
S Cl
Pine tree growing in tube
P XRF and co-localisation XRF maps
Conclusion
Mapping the elements on root sections can enable us to understand better the biological mechanisms sustaining the functioning of the symbiosis. These first characterizations of ECM using synchrotron XRF evidenced, in particular, the role of HcPT2 in increasing P concentration in the xylem.
S Cl
K
XRF spectrum Fit of the XRF spectrum
Profiles across P XRF maps
Using a standard (NIST bovine liver), K and P % were calculated in different cells / compartments.
The presence of the fungus increased always K and P contents of root cells compared to NM roots. Remarkably, OE-PT2 increased specifically the P contents in the central cylinder and in the cytosol of the cortical cells of ECM roots. These results support a role of Hc-PT2 in the unloading machinery of P from the fungal cells in the Hartig net, enabling a higher P content in the xylem towards the shoots.
0 1 2 3 NM Ctrl OE-PT2 Cortex Cc K (%) 0 1 NM Ctrl OE-PT2 Cortex Cc P (%) 0 0,2 0,4 0,6 NM Ctrl OE-PT2 cytosol vacuolar K (%) 0 0,1 0,2 0,3 NM Ctrl OE-PT2 Cytosol Vacuolar P (%) ECM NM
Elemental concentrations calculated from profiles across K and P XRF maps
Interestingly, in mycorrhizal roots (ECM), it was also possible to distinguish the external fungal sheath (FS) and also the fungal cells belonging to the Hartig net (HN).
Plants were grown in tubes for 2 months, whether or not inoculated with Ctrl or OE-PT2 fungal strains. A nutritive solution
containing nitrate (1 mM) and Pi (0.1 mM) was regularly added throughout the culture. Plants were extracted from the tubes and
the root system rinsed in water. Short roots were cut under a stereomicroscope, embedded in a cryoprotectant and immediately
plunged into isopentane cooled by liquid N
2to fix the cells and their contents. Cryo-thin sections of 20 µm were prepared using a
cryotome and analysed at SOLEIL synchrotron on the microfocused LUCIA beamline equipped with a N
2cryostage.
SOLEIL synchrotron is acknowledged for beamtime allocation through proposal 20160232.
P K Cl 200 µm 200 µm P K Cl P
