High-resolution phenotyping Water flow dynamics Chloroplast movement

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Remote sensing – Beyond images
Mexico 14-15 December 2013

The workshop was organized by CIMMYT Global Conservation Agriculture Program (GCAP) and funded by the Bill & Melinda Gates Foundation (BMGF), the Mexican Secretariat of Agriculture, Livestock, Rural Development, Fisheries and Food (SAGARPA), the International Maize and Wheat Improvement Center (CIMMYT), CGIAR Research Program on Maize, the Cereal System Initiative for South Asia (CSISA) and the Sustainable Modernization of the Traditional Agriculture (MasAgro)

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  • Synchrotron X-ray imaging is similar to medical X-ray imaging in allowing visualization of internal structure based on absorption and phase contrast characteristicsHowever, it provides far better temporal and spatial resolution which permits to distinguish individual xylem vessels in a VB.Generally, living tissues and xylem vessels have a similar absorption characteristics as it basically contains mainly water.That’s why refilling dynamics were particularly appropriate.However, it requires differences in absorptionIn terms of dynamics,
  • X-ray image Cross section  3DThe xylem structural specificities were investigated through histological cross sections and SEM imaging of intervessel pit distribution. On the other hand, the use of gold nanoparticles (AuNP) as flow tracers and synchrotron X-ray high resolution CT revealed growth and development dependent water uptake dynamics in individual xylem vessels of each vascular bundle.Vascular tissue within the Arabidopsis thaliana inflorescence stemsSections showing vascular bundles close to the apical meristem (top) and base (bottom) of a single inflorescence stem. Phloem (ph), procambium (pc), protoxylem (px), mature metaxylem (mmx) and developing metaxylem (dmx) are indicated.
  • Fig. 2 Two-photon microscopy (TPM) imaging of maize leaves (a) Large field of view (235×222μm2) TPM imaging of a leaf epidermis showing autofluorescence from stomata and cell wall constituents (λ=820 nm, p=22 mW)(b) TPM imaging (λ=780 nm, p=20 mW) near the leaf surfaceshowing the location of stomata (S) above the mesophyll (M) and fibers above the bundle sheath (BS) located underneath (λ=820 nm, p=22 mW)(c) TPM images of Mchloroplast autofluorescence35 μm below the leaf surface.Dashed circles indicate aggregation of several chloroplasts.(d)3D rendering of subepidermal chloroplasts in intact leaves after 1 hour in the dark(e) 3D rendering of subepidermal chloroplasts in intact leaves after 4 hours of light exposure(f) Reconstitution of chloroplast movement before (in yellow) and after light exposure (in green): red arrows indicate the displacement of each chloroplast center of gravity. (Scale bars=10 μm)
  • High-resolution phenotyping Water flow dynamics Chloroplast movement

    1. 1. High-resolution phenotyping Water flow dynamics Chloroplast movement Hae Koo Kim, Joonghyuk Park, Jeong Eun Ryu, Sungsook Ahn, Ildoo Hwang and Sang Joon Lee
    2. 2. Plants in Environmental & Energy Sciences  Diversity in vascular system: Model plants  Central Role in the Water Cycle on Earth (conifers): water transport over 100m -Gymnosperms  Green revolution yet to come -Angiosperms (monocots & dicots): few cm to tens of m for smallholder farmers?  Importance of the Plants  Agriculture: up to 70% of the Water & mineral transport : Xylem water use  “Blue revolution” in Agriculture: Drought stress in water-limited environments - Drop by Drop - Gene by Gene Pennisi (Science, 2008)  Water transport in plants Cohesion-Tension Theory, Böhm, 1893; Dixon and Joly, 1895) Scholander et al. (1965) Science 148 Johnson & Dixon (1965) Nature 208 Scholander et al.,1965 Balling & Zimmermann, 1990 Pockman et al. (1995) Nature 378 Holbrook et al. (1995) Science 270
    3. 3. Visualization of Water flow at different level on Earth 6,650 km
    4. 4. Synchrotron facilities PAL, Pohang PF, Tsukuba SSRF, Shanghai
    5. 5. Visualization of water flow dynamics using synchrotron X-ray imaging Synchrotron X-ray imaging -Absorption and phase-contrast based imaging -High temporal (ms) and spatial resolution (μm) -Ideal to visualize xylem vessels of monocots -Real-time dynamics of refilling process Kim & Lee, 2010 New Phytologist
    6. 6. Phenotyping Xylem Structure/Function relationships Sap flow dynamics in xylem vessels Xylem Safety Efficiency Structure Fibers Vascular bundle [WATER TRANSPORT] Efficiency vs. Safety trade-off in water transport Function High resistance Modularity Support Repair Parenchyma Vessels Storage Transport Low resistance Connectivity Phloem FUNCTION of Xylem: Efficiency & Safety of water transport STRUCTURAL characteristics of XYLEM  Sap flow dynamics
    7. 7. Efficiency and Safety trade-offs in water conduits Number of pits 16-20 11-15 6-10 1-5 0 Cluster 1 Cluster 2-2 Cluster 2-1 U R 8 2 P, Q R 4 Pc P Cluster 3 8 Metaxylem vessel number and lumen area • Diameter A:B:C=4:2 :1 • Vessel area A : B : C = 16 : 4 : 1 • Flow rates A : B : C = 256 : 16 : 1 Cluster Number (ea) AVG (µm2) MAX (µm2) MIN (µm2) 1 73 226.7 (±102.3) 496.7 66.2 2 83 201.8 (±87.4) 489.6 76.0 3 126 108.7 (±37.5) 318.3 52.9 7
    8. 8. Visualization of internal structure of plants and flow dynamics Kim and Lee., 2010 New Phytologist Ahn et al., 2010 ACS Nano
    9. 9. -Long distance water transport (efficiency) -Short distance, higher resistance, local water distribution (safety)
    10. 10. Synchrotron X-ray CT: Vascular bundle organization in maize leaf
    11. 11. 3D organization of Vascular bundles in maize leaf
    12. 12. Xylem anatomical characteristics • Safety: Regulation of -Axial flow dynamics by perforation plates -Radial flow dynamics by pit membranes & network connexion Basis of embolism, cavitation mechanism Environmental effects: transpiration, osm ostic stress • Safety feature of perforation plates, lateral pits: air bubbles appearance/re moval, direct radial inflow of water  Network • Efficiency: -Effect of enhanced transpiration -Long distance water transport system In planta sap flow tracking Protoxylem (PX) Metaxylem (MX) •Use of various type of particles to investigate xylem cell wall properties (interactions of AuNPs with cell walls)
    13. 13. Flow dynamics in dicot plants (Arabidopsis) of dicot xylem structure Arabidopsis as model Experimental method: -Synchrotron X-ray CT [Xylem network 3D organization] -Hydrophilic AuNP solution [Indicator of sap flow rate] AuNP-OH Xylem SCW HO CH2 CH2 CH2 CH2 OH Difference of xylem vessel activity monitored by AuNP staining profile 13
    14. 14. Pattern of xylem vessel activity in normal condition Normal uptake profile (transpiration driven) Pattern of xylem vessel activity generated by external pressure Artificial uptake profile (external pressure driven)
    15. 15. Visualization of water transport pathways Hydrophilic gold-nanoparticles (AuNP) : tracers of axial & radial water flow pathways c Interestingly, all metaxylem vessels do not show the same level of activity for water transport 17
    16. 16. Regulation of water transport along plant height Differences in vascular bundle organization and pressure gradient directly impact AuNPs staining profile along the height of the inflorescence stem 18
    17. 17. Water flow pathway in the whole plant scale Efficient and safe water flow may coexist with one system as division into long-distance and local distribution 19
    18. 18. Visualization of chloroplast movement using two-photon microscopy Two-photon microscopy Tube lens Scan lens Ti:Sapphire laser Dichroic mirror DM1 Tube Scan lens lens Dichroic mirror DM2 Galvanometer scanner PMT Piezo stage Filter Objective lens Computer Translation stage
    19. 19. Monitoring of chloroplast movement depending on light S S S M BS S S S
    20. 20. Summary Place of plants in Environmental & Energy Sciences Importance of understanding Plant-Water relation Synchrotron X-ray imaging method is a powerful tool to study plant vascular structure and sap flow dynamics -Monocot and dicot plants 3D xylem structure (“Wiring diagram” of plant microfluidic system) -Demand-driven flow system -Efficiency and safety of water transport Perspectives of High-resolution phenotyping -Nano-CT of xylem -Optical Coherance tomography -Two-photon microscopy (photosynthesis)
    21. 21. Acknowledgements Advanced Biomass R&D Center Prof. Sang Joon Lee (Department of Mechanical Engineering, POSTECH) Prof. Il Doo Hwang (Department of Life Sciences, POSTECH) Dr. Sung Sook Ahn Joong Hyuk Park Jeong Eun Ryu Synchrotron facilities PAL, Pohang SSRF, Shanghai PF, Tsukuba

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