1. Plant Biology
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Testing the Münch hypothesis of long distance phloem transport in plants

  1. Michael Knoblauch  Is a corresponding author
  2. Jan Knoblauch
  3. Daniel L Mullendore
  4. Jessica A Savage
  5. Benjamin A Babst
  6. Sierra D Beecher
  7. Adam C Dodgen
  8. Kaare H Jensen
  9. N Michele Holbrook
  1. Washington State University, United States
  2. Harvard University, United States
  3. Arnold Arboretum of Harvard University, United States
  4. Brookhaven National Laboratory, New York
  5. Technical University of Denmark, Denmark
Research Article
Cite this article as: eLife 2016;5:e15341 doi: 10.7554/eLife.15341
6 figures, 1 video and 1 data set


Figure 1 with 1 supplement
Geometrical parameters of a moderate sized morning glory plant.

(A) A 7.5 m long morning glory plant with source leaves along the stem. (B-D) Scanning electron micrographs of sieve plates at 1 m (B), 4 m (C), and 7 m (D) from the base of the stem (as indicated by red arrows in B–D). E–J) Cell geometrical data were collected separately for internal (red) and external (blue) phloem. Average sieve element radius (E; n ≥ 10 per data point), length (F; n ≥ 10 per data point), pore number (G; n ≥ 10 per data point), and pore radius (H; n > 150 per data point) result in a conductivity of ~1 µm2 for the external phloem but an increasing conductivity along the stem for the internal phloem (I). The total phloem area (J) is, however, significantly higher for the external phloem. Error bars show standard deviation. Scale bars in B–D =10 µm

Figure 1—source data 1

Source data of sieve tube geometrical parameters for Figure 1 and Figure 3—figure supplement 3.

Figure 1—figure supplement 1
Stem anatomy of morning glory plants.

(A) Staining of morning glory stem cross sections at 4 m with a mixture of calcofluor white and aniline blue allowed visualization of sieve plates in the bicollateral bundles of external (blue dashed arrow) and internal (green dashed arrow) phloem, which are easily discernible from primary (yellow arrow) and secondary xylem (green arrow) as well as sclerenchyma (white arrow). (B) Internal and external phloem areas are highlighted in white in stem cross sections (from left to right) at 7 m, 4 m, and 1 m from the base of the stem. Scale bars = 1 mm

Figure 2 with 3 supplements
Phloem flow relevant parameters in a medium sized morning glory plant.

An illustration summarizing the findings in a medium sized plant with leaves attached along the entire length of the stem. Cell geometrical data were taken at 1 m, 4 m, and 7 m (blue lines) along the stem. Resulting conductivities are indicated in green. Source sieve tube turgor measurements (red) were taken in the main vein of leaves along the stem axis, sink turgor (black) in root tips, and average flow velocity was measured using 11CO2 labeling (orange).

Figure 2—figure supplement 1
In situ viscosity measurements. 

(A) 2-NDBG is loaded into the phloem in situ and observed by confocal microscopy in the midrib of a mature leaf in morning glory. The image reveals the location of sieve elements (SE), companion cells (CC) sieve plates (solid arrows) and sieve element plastids (dashed arrows). (B) A corresponding color coded fluorescence lifetime map reveals the relatively low viscosity of phloem sap of 1.7 mPas (blue) in contrast to the much higher viscosity above 5 mPas of the cell wall, membrane, and nucleus areas. (C) Calibration curve of 2-NBDG lifetime versus viscosity for aqueous sucrose solutions at temperature T = 298 K. A strong lifetime change between one and 10 mPas renders 2-NDBG a good probe for intracellular viscosity measurements. n ≥ 21 for each data point. Error bars show standard deviation.

Figure 2—figure supplement 2
In situ sieve tube turgor measurements.

Two frames extracted from Video 1 showing in situ pico gauge pressure measurements. A sieve tube (bright green) translocates distantly applied fluorescent dye. The black arrow demarcates a sieve plate. The red arrow points to the location of the water oil interface before impalement into the cell. The turgor pressure of the sieve tube results in a compression of the pico gauge filling oil, indicated by the movement of the meniscus interface (blue arrow). Inflow of fluorescent dye into the water phase of the pico gauge (yellow arrow) provides evidence, that the measurement occured in the sieve tube.

Figure 2—figure supplement 3
Symplastic phloem unloading in the root tip of morning glory plants. 

Carboxyfluorescein-diacetate is a phloem mobile fluorophore that can be loaded into the phloem of a leaf of a morning glory plant. Once the dye enters a cell, the diacetate residue is cleaved and the fluorophore carboxyfluorescein is generated which is membrane impermeant. The dye is then translocated with the phloem sap into sinks. (A,B) Since the dye can only exit cells symplastically, the spread of the dye shown in the fluorescence micrograph (B) within the root tip (A) indicates symplastic unloading.

Figure 3 with 3 supplements
Geometrical parameters of a large morning glory plant with partially defoliated stem.

Geometrical data of a 17.5 m long morning glory plant after 5 months growth with daily removal of developing side branches and flowers as well as removal of source leaves below the top 4 m. (A) Total phloem area at different locations along the shoot. Plotting phloem area versus distance indicates that only the external phloem (blue) increases in area significantly (internal phloem, red). (B–F) Cell geometrical data for sieve element radius (B; n ≥ 10 per data point), sieve element length (C; n ≥ 10 per data point), sieve plate pore number (D; n ≥ 10 per data point), and sieve plate pore radius (E; n ≥ 380 per data point) reveal that sieve tube conductivity (F) increases with the length of the transport pathway. Please see Figure 3—figure supplement 3 for a comparison of the parameters and standard deviations between the moderate sized foliated morning glory (Figure 1) and the partially defoliated large morning glory plant.

Figure 3—source data 1

Source data of sieve tube geometrical parameters for Figure 3 and Figure 3—figure supplement 3.

Figure 3—figure supplement 1
Habitus and anatomy of a partially defoliated large morning glory plant.

17.5 m long morning glory plant (left) after 5 months growth with daily removal of developing side branches and flowers as well as removal of source leafs below the top 4 m. Confocal images of cross sections (right) at the indicated location (in m) from the shoot base with highlighted phloem area. Scale bar = 2 mm.

Figure 3—figure supplement 2
Anatomical adaptation to increase sieve tube conductivity. 

(A) Scanning electron micrograph of a stem cross section at 4 m distance from the stem base of the 17.5 m long morning glory plant, showing the anatomical adaptation of sieve plates to increasing demand by changing plate anatomy from simple to compound with steep plate angles to increase plate surface area (compare Figure 1B–D). (B,C) Confocal micrographs of tangential sections through the phloem at 15 m (B) and 5 m (C) from the base of the stem stained for cellulose (blue) with calcofluor white and callose (red) with aniline blue. Youger tissue in close proximity to sinks has mainly simple sieve plates in a 90 degrees angle, while tissue distantly located from the closest sink generated compound sieve plates in steep angles. Scale bars: A = 50 µm; B,C = 100 µm.

Figure 3—figure supplement 3
Comparison of geometrical parameters between a small foliated (compare Figure 1; green = external phloem, black = internal phloem) and a large, partially defoliated (Figure 3; blue = external, red = internal) morning glory plant.

Error bars show standard deviation. The significantly larger sieve plate pore radius (C) in the external phloem of the large plant results in a several times higher conductivity (E). Changes in other geometrical parameters (A,B,D) have less impact on the tube conductivity. The conductivity is relatively low in the small foliated plant, because conductivity scales as 1/r2 when the SE radius r is large. In combination with an increase in conducting area (F), the bulk of phloem transport during secondary growths occurs through the external phloem.

Figure 4 with 2 supplements
Phloem flow relevant parameters in a morning glory plant with increasing leafless stem length.

An illustration summarizing the findings in large morning glory plants after artificial increase of source-to-sink transport distance achieved through continuous partial defoliation. Leaves were maintained only on the upper four meters of the plant and pressure was measured throughout the growth period. Plants with a short distance between the leaves and the roots maintain a relatively low sieve tube turgor pressure (crimson) in the source phloem in the range of 0.7–0.8 MPa, but the pressure scales with increasing length (black) of defoliated stem and the conductivity increases ~5 fold (green) compared with the 7.5 m long plant shown in Figure 1 and 2. Flow velocity (blue) was measured by 11C application.

Figure 4—figure supplement 1
In situ measurements on large morning glory plants.

(A) In situ turgor measurement using pico gauges and a fixed stage remote controlled fluortescence microscope. (B) A large morning glory plant during Micro PET scan experiment setup. (C) PET scan image of the lowermost leaf provides evidence that transport occurs through the petiole only towards the root. No label was detected in the upper stem towards the shoot tip (location of stem indicated by white lines).

Figure 4—figure supplement 2
Morning glory stem growth.

The helical growth of morning glory stems to wrap around objects in order to climb leads to an increase in the sieve tube length compared to straight stems.

Anatomical adjustments to growth conditions. 

An illustration summarizing the findings in a large morning glory plant with leaves along the length of the stem. In contrast to partially defoliated plants, the conductivity remains relatively low, likely due to the shorter distance from source leaves to sink tissue.

Figure 5—source data 1

Source data of sieve tube geometrical parameters for Figure 5.

Phloem pressure gradients in relation to phloem unloading.

Schematic drawing of a single source leaf (green) loading assimilates into the phloem (blue) and unloading through plasmodesmata (black) into a single root tip (yellow). (A) Independent of the plant size the pressure manifold model proposes nearly uniform high pressure along the stem, but large differences between sieve elements and surrounding cells in the unloading zone where the steepest gradients are found. (B) Our data suggest that tube resistance in the stem of morning glory plants consumes most of the pressure gradient, contrary to the high pressure manifold model.



Video 1
In situ sieve tube turgor pressure measurement using a pico gauge. Real time video of a pico gauge measurement.

The fluorescence of a distantly loaded sieve tube provides evidence for tube intactness and transport. The oil – water interface in the pico gauge moves backwards when the pico gauge is impaled into the sieve tube, indicating compression of the oil volume. Inflow of fluorescent dye into the pico gauge tip provides evidence that the measurement occured in the sieve tube.


Data availability

The following data sets were generated
  1. 1

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