Potential of (portable) NMR and MRI for plant phenotyping. Henk Van As

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1 Potential of (portable) NMR and MRI for plant phenotyping Henk Van As

2 NMR/MRI and plant phenotyping Non-invasive NMR and MRI sensors to measure plant performance and uncover genetic determinants of enhanced agronomic traits. A number of approaches and parameters are available from such sensors to study complex plant physiological traits such as growth, biomass accumulation and yield throughout the plant (crop) growing season. Both below-ground (3D root architecture), above-ground (stem, leaves, water status, growth, fruit development) and transport traits can be studied.

3 Suggested NMR/MRI accessible traits Rascher et al, 211, Functional Plant Biol. 38 There are some more possibilities.

4 (TD-)NMR and MRI NMR: non-spatially resolved, fast, detailed information, relatively cheap. Problem: tissue heterogeneity MRI: spatially resolved, longer measurement times, less detailed information on cell level. Problem: expensive dedicated equipment. But.

5 The (TD-)NMR signal ALL signal amplitude solids liquids solid+ liquid LIQUIDS t= excitation time (ms)

6 TD NMR: dynamics of water 1 H s One multi echoes analysis Sum of exponentials T2 distribution (CONTIN)

7 onion More details: use diffusion of water molecules to characterize free diffusion path length, combine with T2 =4 ms =16 ms =32 ms =128 ms

8 Effect of membrane permeability on T2 Cherry tomatoes dipped in N2 for different times Amplitude R 2 (s -1 ) T 2 (s)=1/r 2 x35. Amplitude 8.5. T 2 (s) 1.4

9 Longest T2, cell (vacuole) size and membrane permeability (H) MRI 1/T 2,obs = H(1/R x +1/R y +1/R z ) + 1/T 2,bulk 1/T 2 (s -1 ) pearl millet (n=4) maize (n=4),5,1,15,2,25 1/R longitudinal + 2/R radial (m -1 ) Cryo-SEM L. van der Weerd et al., Plant, Cell and Environment 22

10 Stem (apex) growth and T 2 during osmotic stress: maize and millet shoot growth (a.u.) PEG 1/T2 (s -1 ) 12 1 Maize Maize Millet no stress 48 h PEG time (hours) pearl millet maize amp 1/T 2 PEG ADC trans ADC long time (h) van der Weerd et al., Plant, Cell and Environment 22

Tonoplast membrane permeability in millet during osmotic stress: 1/T 2,obs = H (S/V) + 1/T 2,bulk H (1-5 m s -1 ) 7 6 5 4 3 Osmotic stress PEG MRI 2 24 48

11 Tonoplast membrane permeability in millet during osmotic stress: 1/T 2,obs = H (S/V) + 1/T 2,bulk H (1-5 m s -1 ) Osmotic stress PEG MRI time (h) Increase in H may facilitate water redistribution between tissues: aquaporins? van der Weerd et al., Plant, Cell and Environment 22 Cryo-SEM

12 T2, diffusion and permeability cytoplasm R y R x Relaxation: 1/T 2,obs = H(S/V) + 1/T 2,bulk Can we get information about S/V? H vacuole Yes! Diffusion as a function of diffusion time: Restricted due to finite compartment size Short diffusion distance: D app (=ADC) = D S/V C D 1/2 1/2 Long time-scale: (tissue) permeability Can we become independent of S/V? Yes: T1/T2

13 Conversion of starch: interaction between mobile and solid protons 2C 4C T = 8 weeks 8 weeks

14 Transport traits: especially appealing Xylem: Simple proportionality (modified Darcy s law) to compare (woody) plants by xylem specific conductivity, K s (length normalized) is rate of sap flow per crosssectional area at a given pressure gradient: K s /(LA/XA) VPD.g s.ht/( s - L ) Phloem: transport between source (leaves) and sinks (roots, fruits, growing tissue,..). Can it limit optimal photosynthesis? Loading from sucrose/starch. Can we benefit from optimized unloading?

Consequently little is known regarding the dynamics of sap flow and conductivity in an intact plant.

15 Integrated model: photosynthesis, water and sugar Xylem and phloem sap flows are difficult to study because of their extreme sensitivity to invasive experimentation. Consequently little is known regarding the dynamics of sap flow and conductivity in an intact plant. If and when xylem transport and phloem can be limiting for optimal photosynthetic activity, CO2 uptake and plant performance in greenhouse conditions are not yet resolved. Phloem??? Xylem? De Schepper and Steppe, J Exp Bot. 21

16 Dynamic or displacement imaging P(r r,), conditional displacement probability displacement r + gradient G => frequency or phase shift diffusion, perfusion random, => no net phase shift flow: equal for all spins => net phase shift detect signal as function of G =FT=> distribution of displacements r

17 Propagator and flow characteristics For every pixel in the image or selected regions! Volume-flow R Q R R max P n R, R A ref P(R) (a.u.) A total propagator flowing water stationary water Average velocity v R R max n R R R max R P P n R, R, R displacement (um) (?m) (m) R FCA R R max P n R, A ( Q / v ) ref

18 Flow: xylem and phloem in Maize Amount of water flow: xylem (day) T2 flow: xylem and phloem (night)

25 c T 1 phloem and xylem volumeflow d 15*1-3

19 NMR/MRI: axial and radial transport and water content in storage pools a water content b T c T 1 phloem and xylem volumeflow d 15*1-3 Resolution: 8-1 m Velocities: 1 m/s tens of cm/s 1 5*1-3

20 Dynamics in xylem and phloem flow a volume flow (mm3/s) water content a Poplar b velocity (mm/s) volume flow (mm3/s) T 2 c Tomato velocity (mm/s) stationary water / pixel (%) a flow conducting area / pixel (%) b 2% 1 c volume flow (mm3/s) b time (h) T Castor bean 1 1 time (h) volume flow velocity velocity (mm/s) d time (h) phloem and xylem volumeflow volume flow (mm3/s) d Tobacco 5*1-3 15* time (h) velocity (mm/s) average linear velocity / pixel (mm/s) c 1% 2.4 volume flow / pixel (mm 3 /s) d 4% 7.5*1-3 (Windt et al, PC&E 26) 4*1-3

21 Dynamics in flow conducting area / conductance!? (Windt et al, PC&E 26) flow conducting area (mm 2 ) linear flow velocity (mm/s) a b Phloem Day Night poplar castor bean tomato tobacco flow conducting area (mm 2 ) linear flow velocity (mm/s) a b Xylem Day Night poplar castor bean tomato tobacco. poplar castor bean tomato tobacco

22 Phloem flow velocity can not simply be explained by conductivity and / or laminar flow? Mullendore et al, The Plant Cell 21

23 Grape: bi-directional Xylem and phloem flow Phloem has been observed in both directions: Upward and downward (different plants)

24 Imaging stem-truss stalk connection lighting system climate chamber A plant iron yoke magnet poles pole caps gradient system B pot electromagnet and open gradient system

25 Flow in truss stalk tomato during 8 weeks of truss growth: Ratio [xylem]/[phloem] is >3:1and not 1:9!!!!back flow III II c b a 5 fruits red, one green remaining I last fruit red volume flow + - Windt et al, Plant Physiol 29

26 Below ground: 3D root anatomy and water uptake Maximum intensity projection 3T-MRI, Fast Spin Echo 128 x 128 x 128 pixels (FOV 1 cm -> ~.8 x.8 x.8 mm 3 per pixel) Total 3D experiment time 51 min 12 s

27 Root transport: e.g. ecotypes in relation to aquaporin expression Arabidopsis thaliana ecotype: Columbia; age: 4~6 weeks.

Transport in roots: pathway and tissue permeability Comp 1 (larger T2 and cell size) - Millet and Mais Comp 2 (smaller T2 and cell size) - Millet and Mais T35 o C T25 o C T15 o C T5 o C T35 o C T25 o

28 Transport in roots: pathway and tissue permeability Comp 1 (larger T2 and cell size) - Millet and Mais Comp 2 (smaller T2 and cell size) - Millet and Mais T35 o C T25 o C T15 o C T5 o C T35 o C T25 o C T15 o C T5 o C Permeability, m/s 1E-4 Permeability, m/s 1E-4 1E-5 1E-5 control stress stress2+gd control stress stress2+gd Pearl Millet Mais control stress stress2+gd control stress stress2+gd Pearl Millet Mais

29 NMR / MRI and phenotyping Anatomy: leaf thickness, 3D root structure, thickness of different tissues, cell size, vessel diameter, compartments, Internal parameters: water content in different tissues, transport and flow conducting area (xylem and phloem; sink-source connection), membrane or tissue permeability, starch content (leaves, beans), Static versus dynamics (growth and adaptation processes and rate)

30 MRI Lab bound, but... starting to become (trans)portable

31 Dedicated Plant MRI systems and climate control:.7 and 3T lights climate chamber plant gradient probe and RF coil electromagnet (.75 T) Growth medium Spiral cooler Balance Cooling unit P.C.

32 Chloroplasts, photosynthesis and single-sided portable NMR = 2 ms = 3 ms = 5 ms = 1 ms

33 Rascher et al Func Plant Biol 211

34 Mobile MRI: Kimura et al, 211

35 Halbach Tree scanner FZJuelich: Blumler et al.

36 Hinged Halbach (NMR-CUFF) and U- shape magnet Windt et al, JMR 211 Rascher et al, T 2.5Kg

37 Tree Hugger An open access transportable 1.1 MHz 1 H MRI system for the in situ analysis of living trees in the forest. Access up to 21 cm. Magnet: 55 kg. Jones et al, JMR 218: (212)

38 Conclusions Large number of parameters, some quite unique, is available by both NMR and MRI. The only systematic application (to my best knowledge) is MRI of 3D root anatomy (24 plants/day, can be increased to max 5 plants per day) There is a strong potential, but protocols and hardware have to be further developed This can only be realized via pilots combining NMR/MRIsts and plant scientists

39 Acknowledgements: Carel Windt Natalia Homan Frank Vergeldt Edo Gerkema, John Philippi Alena Prusova Louise van der Weerd, Tom Scheenen, Timur Sibgatullin NWO, LNV, WUR, SENTER, EU Space for partner logo s (place a white shape behind the logo s to hide this text and border)

NOTES: CH 36 - Transport in Plants

NOTES: CH 36 - Transport in Plants Recall that transport across the cell membrane of plant cells occurs by: -diffusion -facilitated diffusion -osmosis (diffusion of water) -active transport (done by transport