Phloem Translocation

Phloem Translocation

Phloem Organization & Structure

Phloem is one of the two Vascular Tissues in plants. It is almost always associated with Xylem. Xylem conducts water and provides structural support. Phloem is responsible for the translocation of Carbohydrates and some other metabolites. It is important to remember however, that Phloem Sap is mostly Water. The Vascular Tissues are contained in Vascular Bundles for stems and leaves. The bundles usually have Xylem on one side and Phloem on the other. Roots tend to have a core of Xylem with surrounding Phloem.

Vascular Bundles in a Typical Dicot Stem: The Phloem lies between the Xylem and the Fibers which both have cells with thick lignified walls. à à à

Makaloa (Cyperus laevigatus) a Monocot Stem showing many Vascular Bundles distributed throughout the stem diameter

Secondary Phloem is located outside the Secondary Xylem (wood) in woody Stems & Roots. Secondary phloem may contain thick-walled, lignified Fibers. Secondary phloem is often called the "Inner Bark".


		Typical Dicot Leaf: The Vascular Bundles are often difficult to locate because they do not travel at right angles to the plane of cross-sections.
Monocot Leaves: Corn above and Sugarcane (right). Note the Vascular Bundles (veins).

Xylem is easy to spot because it contains cells with thick, lignified walls and large diameters. There is usually a discernable, developmental pattern as well. Phloem cells do not generally have such readily identifiable traits. Consequently, greater scrutiny must be applied to unambiguously identify the Phloem. The Sieve Plate is the most distinctive anatomical trait for Phloem.

Sugarcane Vascular Bundle stained with Toluidine Blue. The Phloem has stained pink. The Sieve Tube Elements (STE) and Companion Cells can be identified based on their size and organizational pattern. The solid pink area in one of the STEs is a Sieve Plate.

Sieve Plate from a commercial slide: The large openings of the Sieve Pores are evident. Sieve Pores allow for cytoplasmic continuity between adjacent STE. Callose is secreted outside the Plasmalemma and lines each Sieve Pore. The size of the pore is regulated by the amount of callose present. Sieve Pores can be completely closed at the end of a growing season or after a wound. The latter is extremely rapid compared to the former.

Phloem can contain the following cell types: Sclerenchyma, Parenchyma, Sieve Tube Elements, Sieve Cells, Companion Cells & Albuminous Cells.

The term Sieve Element refers to Sieve Tube Elements or Sieve Cells. These are the cells that are directly involved with longitudinal transport. Both have Sieve Pores which are enlarged, Plasmodesmata-like channels in the cell wall that are lined with Callose. The amount of Callose controls whether or not the channels are open or closed. Callose stains selectively with Aniline Blue. This can be used to locate Sieve Elements. Furthermore, Aniline Blue has a strong fluorescence when illuminated with UV or Violet light. This further aids in the location of Sieve Elements.

Sieve Tube Elements (STE) have a concentrated assembly of Sieve Pores on their horizontal "end-walls". The latter are called "Sieve Plates". STE form long Sieve Tubes which greatly facilitate longitudinal transport. Sieve Tube Elements also have Sieve Pores on their lateral walls.

Light micrograph of Sieve Tube Elements stained with Aniline Blue. Note the heavy blue stain in the cytoplasm due to the presence of callose. The Sieve plates are also heavily stained.		Fluorescence Micrograph of Sieve Tube Elements stained with Aniline Blue: The Callose in the Sieve Plates and Sieve pores produces a yellow fluorescence.
Sieve Plate seen with Aniline Blue fluorescence		Polar View of a Sieve Plate Stained with Aniline Blue: The Sieve Pores are occluded by Wound Callose!

Longitudinal Sections showing the Sieve Tubes which are composed of Sieve Tube Elements. Sieve Plates occur on the shared end-walls of each Sieve Tube Element. Note the many Sieve Pores on the lateral walls (above).
Sieve Cells have overlapping end-walls that lack sieve plates. This makes them less efficient in long-distance translocation. Sieve Cells are common in Gymnosperms and non-seed plants like ferns. Sieve Tube Elements are characteristic for Angiosperms. The latter show the highest degree of evolution & functional specialization. Their Sieve Pores appear to contain more membranes than STE. This may reduce their transport efficiency.The cytology of Sieve Elements is greatly simplified. They have a Plasmalemma and cytoplasm. However, there is no Vacuole (hence, no Tonoplast). Membrane-bound organelles are few in number and appear weakly functional. There is NO Nucleus and few Ribosomes. There are many Plasmodesmata with adjacent nucleate, vacuolate cells which are physiologically active. Most Dicots and many Monocots have copious amounts of proteins present in the cytoplasm of Sieve Elements. These block sieve pores when the Sieve Elements are ruptured.Companion Cells (CC) develop from the same progenitor as Sieve Tube Elements. They regulate the physiological activity of the Sieve Tube Elements. Albuminous Cells (AC) which have a different progenitor cell than a sieve element, serve the same function as companion cells when Sieve Cells are present. Both CC and AC are nucleate & contain many mitochondria plus other organelles. They are cytoplasmically dense and have small vacuoles. Phloem Parenchyma cells have large Vacuoles. They may interface with Albuminous cells, Companion Cells and Sieve Elements. All of these can be readily distinguished at the EM level. They can sometimes be discerned at the light microscope level as well.
TEM cross-section of Phloem showing a Sieve Tube Element which appears devoid of contents and a Companion Cell which has a Nucleus, Mitochondria and dense Cytoplasm. The square shows a Plasmodesmata between the two cells.	TEM cross-section of Phloem showing a Sieve Tube Element which appears devoid of contents and a Phloem Parenchyma Cell which has a Nucleus, Mitochondria and a large Vacuole.

There are three types of Companion cells in the minor veins of sugar-exporting leaves. "Ordinary" Companion Cells have Plastids with Thylakoids plus a cell wall with a smooth inner surface and FEW Plasmodesmata. "Transfer" Companion Cells are similar but they have extensive wall in-growths which greatly increases the surface area of the Plasmalemma. Phloem Parenchyma can also have extensive wall in-growths like the Transfer CCs. All of these are thought to be involved in absorbing solutes from the APOPLAST.

Transfer Companion Cell (CC) with Sieve Element (SE): Note the many wall in-growths which expand the surface area of the Plasmalemma and Cell Wall (Apoplast). This increases the Apoplastic absorptive capacity.

Intermediary Companion Cell (IC) with Sieve Element (SE): Note the many Plasmodesmata in the cell wall. These increase the potential for Symplastic Transport.

Intermediary (Intermediate) Cells are Companion Cells that are suited for SYMPLASTIC transport of solutes like sugar. These have numerous Plasmodesmata compared to the other two types of Companion Cell. They have poorly developed plastids.

Radioactive Tracer Studies

The use of radioactive tracers showed that it is Phloem which translocates carbohydrates produced by Photosynthesis. Pulse-chase autoradiography studies using radioactive CO2 or radioactive Sucrose, showed that labeled carbohydrates entered the Phloem for redistribution inside the plant. The radioactive label first appears in the Sieve Tube Elements of the Phloem.

Patterns of Phloem Translocation

Sugar transport in the Phloem is NOT regulated by gravity. It is Bi-directional.

There is a general pattern of carbohydrate redistribution from Sources towards Sinks. Sources are carbohydrate exporting structures like mature, photosynthetic leaves. Storage organs like the potato tuber can also be Sources. Sinks are carbohydrate importing structures like immature, nonphotosynthetic leaves. Storage organs can be Sinks or Sources. The storage root of "wild beet" (Beta vulgaris) is a Sink during its first year of existence. However, it becomes a source during its second year when it provides the nutrients for flowering stem production.

Actively growing Shoot & Root Tips are powerful Sinks. Developing Ovules & Fruits are also strong Sinks. There are several major structural factors which regulate Source-Sink activity.


Ovules become the Seeds in mature Fruits.	The Green Fluorescence seen in these Ovules indicates the Import of sugar.

Proximity of the source and Sink is an important regulating factor. The uppermost functional leaves on a stem provide the carbohydrates for the growing shoot tip. More basal source leaves provide sugars for more basal organs like the roots.

The Developmental Stage of an organ can regulate its strength. Young stem tips are strong Sinks. Quiescent stem tips are not strong sinks. Young Fruits are Strong Sinks while mature fruits are not. Young leaves are strong Sinks while mature leaves are strong Sources. Senescent leaves are no longer strong carbohydrate sources. Dicot Leaves start to become sources when they have reached 25% of their final size. The tips are the first region to export carbohydrates. Eventually, the entire leaf is involved in carbohydrate export.

Vascular Connections between Sources and Sinks facilitate direct translocation. Leaves that have longitudinal vascular connections are said to share an Orthostichy. Such leaves are more readily capable of shipping and receiving carbohydrates from one another than with leaves that are on a different orhhostichy.

Direct vascular connections are not a prerequisite for carbohydrate movement from one part of the plant to another. Vascular interconnections in stems can be very intricate, such that photosynthate from leaves on different orthostichies can be shared. However, the degree of translocation flexibility depends on the species. Some, like Soybean, are very flexible while others are not.

This illustration shows two young Arabadopsis Sink Leaves. The yellow fluorescence indicates carbohydrates in the Phloem. The youngest leaf shows diffuse fluorescence. This signifies Phloem Unloading into immature mesophyll cells. This is strongest at the base of the leaf. The fluorescence is confined to the veins in the upper part of the older leaf. This indicates phloem Loading & Export. The diffuse fluorescence at the base of the leaf shows that unloading is still occurring there. It is customary for leaf tips to become sugar exporters while the base of the leaf is still importing sugar.

Fully Mature Leaf: Note that the fluorescence is confined to the veins. All parts of this leaf are Loading& Exporting sugar.

The Red Fluorescence is due to the presence of Chlorophyll in all of these images.

Phloem Contents

Sucrose is the most commonly found carbohydrate in Phloem. It has a concentration range of 0.3 to 1 M. Nitrogen is generally translocated in the form of Glutamate or Glutamine; Aspartate or Asparagine. These have a low concentration compared to carbohydrates.

Most Plant Hormones (Growth Regulators) are transported in the Phloem. Long-distance auxin transport, especially polar transport, also occurs in the xylem.

Some proteins can be present in Phloem Sap. This includes P-protein which is found in virtually all dicots and many monocots, as well as some enzymes. Phloem tends to have a BASIC pH! The Inorganic solutes, Potassium, Magnesium, Phospahate and Chloride are found in the Phloem. However, Nitrate, Calcium, Sulfur and Iron may be Excluded!

Substance

Composition of White Lupine Xylem & Phloem Sap

Xylem Sap (mg l^-1)	Phloem Sap (mg l^-1)
Sucrose	*	154,000
Amino acids	700	13,000
Potassium	90	1,540
Sodium	60	120
Magnesium	27	85
Calcium	17	21
Iron	1.8	9.8
Manganese	0.6	1.4
Zinc	0.4	5.8
Copper	T	0.4
Nitrate	10	*
pH	6.3	7.9

Various methods have been used to collect phloem sap. One direct method involved the use of Aphids. These are small insects which inject their stylet into Sieve Elements. The positive pressure inside the Sieve Elements pumps the sap into the Aphids who secrete drops of sucrose-rich "honeydew" from their posterior. If the aphids are carefully separated from their stylets, the phloem sap will continue to flow. It can be collected and analyzed. These and other results confirm that Sucrose is the most commonly translocated form of sugar.

Aphid feeding on the Phloem & showing its "honeydew' secretion.

Isolated Aphid Stylet with a drop of Phloem Sap Exudate.

Rates of Translocation

This can be measured as Velocity (distance traveled/unit time) or Mass Transfer rate (quantity passing a cross-sectional area/unit time). Mass transfer rates based on the cross-sectional diameter of Sieve Elements is the preferred method for reporting translocation rates.

The preferred units are meters (m) or millimeters (mm), seconds (s) & kilograms (k). average Velocities = 1 m/hr. The range is from 0.3 - 1.5 m/hr.

These rates are GREATER than the rate of Diffusion!

Phloem Loading

Translocation from the Mesophyll Chloroplasts to the Sieve Elements is called Short Distance Transport

Path = Mesophyll Cell Chloroplast Stroma -> Cytoplasm -> Symplast or Plasmalemma -> Apoplast -> Small Vein (This distance is usually around TWO Cells)

Sieve Element Loading: Sugars are concentrated in Companion Cells and Sieve Elements which form a functional unit.

Export is carbohydrate transport from the loading site to the Sink. This can be called Long Distance Transport.

Short Distance Transport can occur via the Symplast or Apoplast!

Apoplastic Transport

If Apoplastic transport occurs the following should be observed.

1] Transport sugars should be found in the Apoplast.

2] Apoplastically supplied transport sugars should be accumulated by the Sieve Elements.

3] Inhibition of Sugar uptake from the Apoplast should inhibit carbohydrate export.

4] Sucrose uptake by the Sieve Element Complex should require metabolic energy. The Solute Energy of a Sieve Element can be more than twice as much as that of the Mesophyll (-3.0 MPa vs -1.3 MPa). Such a concentration difference can occur only if energy is used.

When these hypotheses were tested they proved to be valid in many cases! Therefore, short-distance transport via the Apoplast does occur in some species.

Sieve Element Loading uses a Sucrose H⁺ Symport mechanism. Proton Pumps (H⁺ ATPases are found in the Plasmalemma of Companion Cells. Protons are pumped into the Apoplast. When their concentration is great enough they are co-transported across the Plasmalemma with Sucrose. This equilibrates the pH gradient and concentrates Sucrose inside the Companion Cells. This process is regulated by the Turgor Pressure and Solute Concentration of the Sieve Elements. A decrease in solute concentration or Turgor Pressure in the Sieve Elements would lead to more Sucrose Uptake.Symplastic Carbohydrate Short-distance transport occurs in some species. The apoplastic scheme presented above occurs with species that use sucrose as their transport molecule and have Ordinary or Transfer Companion Cells. The latter have few Plasmodesmata.

Cross-Section of a Sugarcane Leaf showing the small area used to illustrate Short-Distance Transport & Sieve Element Loading Paths illustrated on the right. The red boxes signify the approximate area & size of the cells involved in the illustration on the right.	This diagram illustrates the Apoplastic (Cell Wall) and Symplastic routes for sugar transport within the mesophyll, Bundle Sheath and Companion Cell-Sieve tube Element Complex. The Apoplastic path is traced with a RED line while the Symplastic route is traced in BLUE.

Diagrams of Sugarcane Leaf Cross Sections: The diagram on the left shows that there are three sizes of Vascular Bundles. In all cases, the photosynthetic Mesophyll is one to 2 cells thick. These interface with a photosynthetic Bundle Sheath that is once cell thick. There may be a layer of Parenchyma or Fibers between the Bundle Sheath and the Phloem. Parenchyma is present in young, exporting leaves while Sclerenchyma is present in older, less productive leaves.

Some species (Sugarcane) use Raffinose (Sucrose + Galactose) and/or Stachyose (Sucrose + 2 Galactose) as transfer carbohydrates and have Intermediate Transfer Cells in their minor veins. These exhibit Symplastic Short-distance Transport. Some examples include Cucumber and Coleus. There must be requisite Plasmodesmatal connections between all the cells involved in order for such a scheme to work. Fluorescent Dye studies have shown this to be true for some species. Agents that block Symplastic phloem loading do not stop this process in some cases. Sugars produced as a result of carbon fixation do not pass into the Apoplast in some species. It is likely that some plants can utilize both types of phloem loading.

The importance of Plasmodesmatal connections and Symplastic transport between the Mesophyll and Bundle Sheath Cells in C4 Photosynthesis is well understood. CO2 is initially fixed in the Mesophyll and is shuttled into the Bundle Sheath cells via Plasmodesmata. CO2 is released and re-fixed in the Bundle Sheath Cells. Photosynthate is translocated from the Bundle Sheath cells to the Companion Cell-Sieve Tube Element Complex. Sugarcane is a C4 plant and it is used above to illustrate the potential path for Symplastic phloem loading.

The concentration of transport sugars in Phloem Sap is higher than in surrounding cells. Furthermore, specific molecules are concentrated in the Sieve Elements. Membrane Transport proteins are highly specific. Consequently it is easy to envision how Apoplast à Symplast loading could lead to a concentration of specific carbohydrates in the Sieve Elements. However, it is difficult to imagine how this occurs with Symplastic Phloem Loading because there is no intervening, selective membrane in the pathway. This could happen if NON-Transport Sugars are specifically converted into Transport Sugars within the Companion Cell Sieve Tube Member Complex. This has been called the Polymer-Trapping Model.

Sucrose diffuses from the Bundle Sheath cells into the Intermediary Cells where it is converted into Raffinose & Stachyose. This lowers the Sucrose concentration. It also lowers the number of osmotically active molecules in solution. Consequently, more Sucrose diffuses into the Intermediary Cells. Raffinose and Stachyose are too large to diffuse into the Bundle Sheath cells. They move to the Sieve Tube Members for long-distance transport.

Model of Sucrose Uptake by Companion Cells: See the Explanation Above.

PhloemLoad2-600.jpg (121474 bytes)

Model of how Polymer Trapping May work

Family Connections

The frequency of Plasmodesmatal connections between phloem cells and other cells with immediate contact has a genetic basis and is strongly associated with certain families. There are also certain growth forms associated with Symplastic or apoplastic phloem loading. Species with lots of Plasmodesmata are the minority and may only represent 10% of all dicots. Consequently, Apoplastic transport is the norm!

Tree, shrubs and vines tend to have a higher proportion of species with evidence of Symplastic transport, while herbaceous plants tend to use apoplastic transport. Species with Symplastic transport are clustered in the Tropics and sub-tropics. One hypothesis is that apoplastic phloem loading is an adaptation to cold temperatures and water stress which inhibit Symplastic transport and are more of a problem in temperate regions. The exact Significance of these observations is uncertain, however, and there is much to be learned before definite conclusions can be drawn.

Phloem Unloading (Import)

This is basically the reverse of Phloem Loading. It has the following major steps. Sieve Element Unloading à Sugars move from Companion Cell Sieve Element Complex to adjacent cells. Short-distance Transport à Movement of sugars to cells in the Sink. Storage & Metabolism à Sugars are metabolized or stored. Sink anatomy Varies more than Source anatomy. Consequently, there is no single anatomical framework for Sinks. Unloading can be Symplastic or Apoplastic. Surprise, surprise! Symplastic unloading is characteristic for Dicot leaves but not for Monocots.

The meristems and elongation zones of Root Tips are also characterized by Symplastic unloading. However, Apoplastic unloading also occurs.

Arabadopsis Root Tips seen with different excitation wavelengths: The image on the right shows a root tip seen with normal microscopic illumination. The other two are seen with wavelengths that elicit fluorescence from a phloem transport reporter molecule. Carbohydrates are brought to the root tip in the central Phloem. The delocalized fluorescence in the meristematic region shows that the carbohydrate is distributed to the actively dividing cells of the meristem which constitute a strong Sink.

Apoplastic Unloading is a necessity in some Seeds because there are NO Symplastic intercellular connection (Plasmodesmata) between the Embryo and surrounding tissues. Sugars first accumulate in the Apoplast of Sugarcane stems prior to their concentration in storage parenchyma cells. Sucrose may be absorbed directly or it may be hydrolyzed into glucose & fructose prior to absorption.

Nearly Mature Seed from Capsella: Note the space (Apoplast) that exists between the Embryo and its surrounding tissue (Endosperm).

The unloading and uptake of sucrose requires the expenditure of energy for this enzymatic reaction and for subsequent steps that lead to its entry into the cell and its organelles. Sucrose transport across membranes involves specific membrane proteins which require ATP in order to work.

The Pressure Flow Model for Phloem Translocation

Diffusion is FAR too slow to achieve the transport velocities observed in Phloem.

The rate of Phloem Transport is around 1 meter/hr.

The rate of diffusion is 1 meter/32 years!

The basic hypothesis is that a pressure gradient exists between sources and sinks that are linked by the Phloem. Water in the phloem moves down the pressure gradient from high pressure towards low pressure just like a garden hose. The sugars and other solutes in the Sieve Elements move by "Bulk Flow" which is exactly what happens in a hose.

SOURCE

Sugars are actively accumulated in the Source Sieve Elements.

This lowers the Water Potential of these cells.

Water enters from the surrounding cells.

This causes an increase in Turgor Pressure of the Source Sieve Elements!

SINK

Sugars are actively removed from the Sieve Elements.

This raises the Water Potential of these cells.

Water leaves the Sieve Elements and the Turgor Pressure Decreases.

Phloem Sap flows from areas of High Pressure to areas of Low Pressure!

Measured Pressure differences between Sieve Elements in Source and Sink Phloem are sufficient for the Pressure Flow Hypothesis to be valid.

Recently, it has been possible to observe the flow of Phloem sap in living plants. This showed that the pores in the Sieve plates were open and that translocation was directional.

This was accomplished by Aart van Bel & Colleagues in Germany. They surgically created two areas in the Petiole of Vica faba leaf. They were able to add dyes to the leaves and observe their flow through undamaged Sieve Tube Elements.

This diagram shows the two dissected "windows' through which Dyes could be added and the living phloem could be observed.
These show a green-fluorescent dye that is moving through the Sieve Elements (SE). SP=Sieve Plate; CC=Companion Cell
The black & white photo on the right provides an excellent view of the Sieve Plate which shows that the Sieve pores are open during translocation.

Model of the Pressure Flow Hypothesis