# 9. Phloem transport mechanisms - some basic considerations  


Transport in action!

The image on the right was made using two filter sets to distinguish the movement of 5,6-carboxyfluorescine (5,6-CF) , a compound that is translocated symplasmically, and  apoplasmically-transported Texas Red - Dextran (3000 Da). A cut leaf was allowed to take up the 5,6-CF which was then chased with a 30 min exposure to a Texas red solution. Sections were cut, and examined using a fluorescence microscope to image the fluorochromes. The process involved here is that a 'sandwich' was made of the two fluorescence images, and the resulting image, shown here shows  the localization of the 5,6-CF (green fluorescence) principally associated with the phloem tissue. The red areas, show localization of the Texas red stain, which is principally associated with the xylem (X) as well as the fibers at the top (adaxial) and bottom (abaxial) sides of this vascular bundle. Texas red is confined to the cell walls and 5,6-carboxyfluorescine is contained within the cytoplasm of living cells.

Did you know that:

  • Solutes move from source to sink?  Why is this so?

  • That sinks may be local or distant? How come?

  • That sink strength is a contributing (perhaps even a controlling) factor in the regulation of transport capacity?  Why is this? Explain.

  • That the loading and unloading system could involve a symplasmic, apoplasmic or mixed mode pathway?

 It is important to distinguish between

  1.  Phloem loading mechanisms

  2.  Phloem transport mechanisms

  3.  Phloem unloading mechanisms

The loading process:

Essentially, can follow either a passive pathway, or could involve an active (accumulating) step.

In the first instance, there may be no energy or thermodynamic demands placed upon the system.

In the second, ATP & NADPH would be needed directly to drive co-transport across membranes.

  The transport process:

  • Phloem transport can be viewed as an entirely passive process, that makes no demands upon the energy cycles of the plant other than energy required for the maintenance of plant membranes.

  • If transport is passive then one could envisage an entirely bulk flow system, driven by concentration gradients established and maintained between the source and the sink. Transport would thus be along or down a concentration gradient.

  • If transport is passive, then metabolic inhibitors would and should have no effect upon the process.

  • Alternatively one could argue that phloem transport is an active process, and one requiring energy (physiological or thermodynamic) in order to drive and maintain it. Here one would envisage ATP NADPH or H+ K+ ion exchange as the driving force.

  • Metabolic inhibitors would severely impede transport, and a velocity decrease would be measurable.

  • In any event, there is little argument that some energy has to be expended upon the way- else a “leaky” system would develop, in which solute loss leads to Ψp and hence, turgor-related changes.

   The unloading process:

Essentially, this must be the opposite of the loading process, as conversion of the soluble carbohydrate into a less osmotically-active form will be necessary in order to set up the driving force of the unloading process.

 Conversion of sucrose to starch reduces the Yp by approximately 50%.

 The process that are involved could be entirely symplastic, mixed symplastic-apoplasmic or apoplasmic at the terminal sinks (such as in parenchyma surrounding and beyond terminal protophloem in shoot and root apices).

What about mechanisms ?

 Are these simple or complex? These can be placed in one of two categories.

  1. Those in which OSMOTIC POTENTIAL is the driving force

 2. Those where ENERGY TRANSFORMATIONS are necessary

We should also distinguish between loading, transport and unloading parenchyma and the sieve element-companion cell complex, at all times, as these regions have differing influences.


An overview of phloem translocation  

The flow of sap and the distribution of foods in plants has presented one of the most demanding and perplexing problems with which botanists have been faced for the past one hundred years or so. The problem has been tackled by many plant physiologists during the 19th and 20th centuries-- amongst these Hales, Hartig, von Mohl, Nageli, Sachs, and Strassburger; and more recently by Dixon, Curtis, Mason and Maskell, Munch, Thaine, Canny Crafts, Crisp, Cronshaw, Esau, Spanner, Eschrich, Evert and van Bel to name but a few.

During the past fifty years or so great strides were made in the development of the ideas and theories on phloem translocation. Some of these theories are based on a fairly simple mass flow mechanism, whilst others, are based on a more elaborate mechanism, or combination of mechanisms.

The great progress which has been made, is due to research in three major areas. Firstly, a study of the anatomy and ultrastructure of the phloem tissue in Gymnosperm, and Angiosperm material. Great progress has been made in the betterment of the embedding techniques, which in turn, meant better cell preservation and less and less unacceptable plasmolysis of the difficult to fix phloem tissues. This has been necessary, due to the ever-increasing use of the transmission electron microscope, and the quest for higher resolution of the particles and components of the living phloem cells. Better fixation has been achieved by using more powerful fixatives, such as glutaraldehyde, in combination with osmium tetroxide, a powerful oxidant, as a post-fixative. Both these fixatives, are applied in buffers, usually 0,05 to 0,1 molar and at neutral or slightly basic pH.

Secondly, Many studies have been performed using aphids and other sap-suckers, in an effort to determine the rate at which substances move in an otherwise undisturbed conduit. Velocities have been calculated, the normal inorganic and organic composition of the phloem has been determined for a great many species using aphids and severed aphid mouthparts. Many experiments have been conducted to determine the direction and velocity of the flow. A number of eminent scientists, and their students, have thus contributed greatly in this area, notably Weatherly, Peel, Eschrich and Evert. It was Eschrich for example, who first postulated that sub- stances could move in opposite directions within the same sieve tube, or within the same file of sieve tubes This was achieved by application   of 14C-Urea acropetally to some feeding aphids, and by the simultaneous application of fluorescein below the feeding aphids. Evert is accredited with the first electron micrographs showing aphid mouthparts in the sieve tubes of barley. The aphid in question was R. maidis . Of course, the most famous micrograph of a feeding aphid is that by Martin Zimmermann, which was the subject of a paper in Scientific American. The aphid, Tuberlolachnus salignus is however a giant compared to some of the others. Those on the Linden trees at Madison Campus, easily reach 6 -1 0 mm in length. In contrast, R. maidis and the one used frequently in our studies such as Aphis nerii are about 1,5 to 2mm in length. The stylets too of T. salignus are like a 1Ocm nail compared to the needle-like stylets of R. maidis or Aphis nerii!

Thirdly, and possibly least successful, have been experiments which have been based upon tracer studies. Why least successful you might ask? Simply is is because early studies relied on water-based photographic emulsions, which were placed in the microautoradiographs of sections of the stem, leaf or root material which had been fed a radiotracer. Later research in the Eschrich and Evert groups, resulted in better fixation and better tracer retention.

Scientific progress is thus a slow and sometimes painful experience -- it is now more than one hundred and fifty years since Hartig (1837) first recognized and associated phloem transport with sieve-elements -- the cells through which all the cellular foodstuffs are transported throughout the plant. Since then, much effort has been ex- pended in the elucidation of their structure and their mode of function in the plant. There is possibly no other tissue in plants, which has caused such heated debate, discussion, even open animosity between the various supporters of the various theories, which have been presented over this time, for the mechanism of phloem translocation.

The companion cell

More and more, the evidence is clear, that the companion cell has a rather important but as yet not fully understood role in this transport system. As mentioned, it is clearly defined for the dicotyledonous plant, and can be easily recognized at the light and TEM levels.

Monocotyledonous plants on the other hand, present a new set of problems as these cells are not so easily recognized. It is clear that the companion cell and its counterpart in the Gymnosperms, must supply the metabolic energy to maintain the metabolic integrity of the sieve tube and sieve cells in dicots and monocots respectively. Add to this, the recent findings based on plasmolytic and plasmodesmatal frequency studies, and a new clearer picture emerges concerning the possible mechanism involved in the two phases of phloem transport, namely short-distance (from the mesophyll to the sieve tube) and long-distance (from source sieve tubes to the various sinks) translocation. Clearly, osmotic forces come into play in this transport system, but there is no denying the fact that there is a distinct need for quite considerable metabolic energy to maintain integrity and possibly,  to assist with the loading process itself also.

Structure-function relationships

That the growth of plants and their normal functioning requires the integrated action of a variety of processes -- be they weeds, crop plants, or merely ornamentals. It is clear that the Absorption of water through roots, the adsorption of nutrients and mineral elements by roots, as well as the biochemical processes of photosynthesis and respiration are elementary to the normal functioning of our plant. Add to this other factors, such as water stress due to excessive water loss and a new gamut of factors emerges, including the fact that various hormone levels can apparently be triggered by such stresses.

The transport of the various food substances from a region of manufacture (source) to a region of biochemical utilization (sink) is thus an essential process which all plants must complete successfully if they are to increase there biomass significantly.

  The symplasm-apoplasm concept revisited

The compartmentation of living cells and the resulting formation of the apoplast and its contained symplast means that intercellular communication requires either diffusion, energy-dependent processes or plasmodesmata to take place. The total mass of the living cells in a plant represent a continuum in which the individual protoplasts being interconnected vial plasmodesmata. Because the plasmalemma is delimited by  the plasmamembrane, the cytoplasm of adjacent cells must be connected through plasmodesmata. As such, the cytoplasm of living cells are thus inter- connected, forming a continuum. The tonoplast is the limiting membrane which separates the cytoplasm from the vacuole and it thus follows that the vacuoles are individual, and do not directly form part of the cytoplasmic continuum. Thus ions taken up by the roots may be able to migrate from cell to cell, without having to cross a permeability barrier anywhere along its chosen route. Movement from cell to cell can, in this simple example, be considered to be based upon diffusion. The process can be accelerated by protoplasmic streaming. Cells which are interconnected by plasmodesmata effectively form domains in which diffusion of material may occur, through plasmodesmata. Taken together, these domains form the sum of the total living protoplasm.

The apoplasm in contrast, must therefore be the sum of all the non-living cell wall continuum of the plant. It constitutes a continuous, permeable system, though which substances such as water and solutes may move freely. All ions and molecules, which enter the root, in effect travel throughout the plant, via the symplast (remember, the xylem constitutes the largest apoplasmic pathway). All substances applied to leaves or stems must diffuse through the cuticle, pass along the cell walls and either move with the transpiration stream to the leaf margins (as do calcium ions and various urea or triazine-based compounds), enter the symplasm and build up in the vacuoles, or migrate to the phloem to be translocated to other parts of the plant. Some substances will enter the leaf cells, but will not translocate (dinitro, pentachlorophenol, and diquat for example) - these may be toxic, and will cause the death of the plant. Some (e.g. phenoxy compounds and P04 3-) will enter and be translocated to other parts of the plant. Others will enter freely, and be translocated via symplast or apoplast (TBA (2,3,6 trichlorobenzoic acid), picloram).

 It has been proposed that the sieve tube constitutes a highly specialized phase of the symplastic transport pathway in plants. The principal features of their structural and functional specialization are the loss of nuclei at maturity, their continuity, loss of tonoplast, parietal cytoplasm and other plastids. The xylem on the other hand, may be considered to be a specialized phase of the apoplast, allowing for the rapid, long-distance transport of water and dissolved inorganic and organic solutes, from roots to shoots.. In its specialization, we witness the fact that the conduits have lost their protoplasts at maturity, and that the end walls become highly specialized - in fact, most of the wall is hydrolyzed, to allow for unimpeded flow.

One should bear in mind the close spatial relationship between the xylem and phloem throughout the symplasm /apoplasm of the living plant. One should also bear in mind the fact that the functioning of the phloem is de- pendent on an adequate supply of water. This water in turn, will affect the osmotic potential of the contents of the sieve tubes, driving up the pressure, and forcing the accumulated solutes to move away, carried by the solvent.

Structure of the phloem

In order to gain a clearer understanding of the phloem, it is necessary to integrate the structure of the phloem with its function. We have already discussed the ontogeny and fine structure of the phloem tissue in higher plants and it is therefore not necessary to repeat this here. You should however, refer back to your notes and Esau, for the account of ontogeny, and structure of the mature phloem tissue. Ensure that you are thoroughly familiar with the changes that take place during the maturation process.

Briefly, the changes that take place involve the loss of the nucleus, degradation of the plasmalemma, degradation of the plastids, including most (if not all) of the mitochondria and the parietal disposition of the cytoplasm. Also, and importantly, is the differentiation of the sieve plate and lateral sieve area pores between concomitant sieve tube members.

Many ontogenetic studies have been carried out over the past thirty years, of which the serious student should refer to those by Esau, Cronshaw, Evert, as well as work by their students and colleagues.

Slime, or p-protein.

The word slime, has come through from the earliest literature of phloem anatomy. Slime bodies, or slime drops are organelles which are commonly found in young, differentiating sieve tube members. These break down during the maturation process, and constitute the slime plugs associated with the sieve plate pores and which are so often referred to in the literature. Most of the early work on slime bodies and slime plugs was undertaken by Esau, Evert and co-workers, using plants such as Cucurbita pepo, as this plant has large-diameter sieve tubes, which can be easily seen at the light microscope level.

Plasmatic filaments.

An internal system of strands or filaments has been in question for a long period of time. In 1932 Alden Crafts discussed the possible existence of such structures, and pointed out the difficulty of demonstrating and photo- graphing them. This difficulty exists today, as the electron microscope does not reveal such structures as being present in the mature sieve elements -- this has however, not daunted those who feel that the mechanism of phloem transport, in some way, involves these non-existent tubular units, or filaments!

There are so-called “plasmatic filaments" present in only some species, but these filaments, if present, are only about 9 - 15 nm in diameter. These filaments have been reported in a number of species of Dioscorea, in Pisum sativum, Primula obconica, Cuscuta, Cucumis (Cucumber) Cucurbita pepo (pumpkin) and in Nymphoides peltata.

Weatherley and Johnson (1968) gave an excellent discussion of the existence of plasmatic filaments, and to their possible involvement in long-distance transport in plants. Various authors gave excellent accounts based on electron microscope of crystalline fibrils, which may be the same as the plasmatic filaments, however, it is clear that it is difficult to visualize just how these filaments could conceivably act as "pumps" as mooted by Alden Grafts in his book entitled "Phloem Transport in Plants". As an alternative, many electron microscopists believe that these crystalline bodies or filaments, are fixation artifacts, produced by the organization of ordinarily soluble proteins, polypeptides and amino acids into reticulate structures. This explanation is acceptable to many who argue that these filaments are derived from the slime bodies.

The path of translocation

The source

Early work on phloem transport was largely concerned with the mechanism responsible for solute movement. Mason and Maskell's important early research showed however, that regardless of the mechanism of transport, that movement of assimilated material takes place from assimilating green tissues, to non-green, growing, and actively respiring tissues in the plant. In short, that the movement follows a source to sink pattern cannot be argued. Munch (1930) presented a simple hypothesis and a simple diagram, which illustrated his concept of phloem transport in plants, in which osmotic forces alone, could account for the movement from source to sink. It is interesting to note that this method of phloem translocation, formulated long before the advent of sophisticated equipment, was ridiculed and argued against for at least forty years. Many researchers came up with reasons just why the Munch hypothesis was unacceptable.

  Composition of the phloem exudate.

As mentioned, the exudate of phloem has been examined from many economically important plants, as well as a large number of ornamentals and weed species. It is fair to say that the average dry matter content of phloem exudate is roughly 10-25%. Of this, roughly 90% appears to be sugar, in one form or another. Amino acids may make up about 0,5% of the exudate in the summer months, and considerably more in the autumn months. According to Crafts and Crisp, there is a general paucity of enzymes, with the exception of the alkaline and acid phosphatases. These enzymes are postulated to play a role in the translocation process, perhaps by assisting in membrane maintenance, or perhaps with the transport process itself. Evidence for phosphatases is not circumstantial, in that they have been detected for some time now at the electron microscope level. The techniques, however, have improved over the past 10 years; as we come to realize that the Gomori technique, which utilizes the localization of a lead salt, is fought with difficulties, and may in many instances, induce misleading results.

Martin Zimmermann has conducted some elegantly simple experiments using Fraxinus americanus. What Zimmermann did, was to essentially incise the phloem of this species, with one incision above the other. Exudation occurred from both incisions, which clearly indicated that the sap above the lower incision was able to move upwards, and exuded from the upper cut. Note however, that this does not preclude losses occurring in a normal basipetal direction. A series of samples were taken of the sap from both incisions. What was recorded was the fact that the concentration of the sugars in the sap decreased steadily with time, which indicated that, the sap was undergoing a gradual osmotic dilution.

The concentration of the sap from the upper incision was slightly higher than that from the lower, indicating that there was still some downward mass flow. The incision however, constitutes a severe injury, and it cannot be precluded that the phloem would have reacted to this state of affairs, by the induction of wound callose. Martin Zimmermann and his group remained fairly active in the field of phloem research up until the early 80's. Many other workers have interested themselves in the collection and analysis of phloem exudate, from plants ranging from the giant kelp, Macrocystus pyrifera, to the giant Metasequoia sempivirens of the California coast. In all instances, the phloem exudate consists mostly of sugar on a dry weight basis.

There is no denying that the severed aphid mouthpart technique is a good experimental protocol. Mittler in 1958, and subsequently used by Weatherly and his co-workers, in fact provide more reliable data in that the plant is not subjected to immediate and severe stresses, as would be the case due to severing of the phloem, by means of incisions. Mittler conclusively demonstrated that the sugars present in the aphid's honeydew, did not differ significantly from that which was present on the phloem sap - in other words, that the aphids merely concentrated the sugars, and excreted them, without changing them materially, or metabolizing them. Experiments conducted by Weatherly and his associates have provided us with much useful information as to the nature of the exudate from the phloem. Most of this research was conducted using Salix (the weeping Willow). Their work confirmed that sucrose was the principle sugar present, but that raffinose also occurred in amounts of up to 15% of the total carbohydrate. During the autumn to spring months, 1O amino acids were identified but only glutamine, aspartic acid, and asparagine were present during the summer months. As much as 2% based on a weight / volume ratio of K+ (potassium) was present, but that the K+ ions were balanced by citric, tartaric and oxalic acids. Subsequently Peel (1963) found that the K concentration in aphid honeydew could contain between 5.2 to 6.9% of the total dry matter. Perfusion experiments (i.e., where the cut ends of the stems were placed in solutions with various ions) indicated that perfusion of the xylem, resulted in a concomitant increase in the concentration of these ions in the phloem. This clearly indicates that the ions, which are taken up by the apoplastic (xylem) stream, enter the symplastic (phloem) system at some point fairly freely. Remember, that I pointed out that certain ions, and or molecules, are can enter the symplastic system more freely than others. It would seem that the naturally-occurring elements, are capable of being transported in either system. This is strong evidence for nutrient cycling in plants, and that the two transport systems, the xylem and the phloem, work in close co-operation to achieve this end.

Obviously, there are many many examples which I could draw your attention to - but one argues that this is not strictly necessary, as all this would achieve, is to add greatly to the evidence, which is clearly not circumstantial. Suffice it to say, that exudate experiments, have clearly indicated that there are a great many phloem- mobile compounds, but that sugar, in the form of sucrose, in the major component of the sap itself.

  Exudation and the effect of applied hydrostatic or osmotic pressure.

Weatherley (see literature cited) demonstrated that the rate of exudation from severed aphid mouthparts was highest during the light periods and lowest during the dark, indicating a direct relation- ship of the light phase and the effect of this light failing on the leaves. When water was forced into the xylem at the cut end of the 15cm stem, then the hydrostatic pressure was raised within the stem, and as a result, the rate of exudation increased above that obtained without increasing the hydrostatic pressure. Weatherly assumed that the mechanism of transport was a pressure-related one, and calculated that the phloem of Bah would require an hydrostatic pressure of 0,6 atm/meter, to account for the then known rates of translocation, assuming that there were no sieve plates. An additional 0,4 atm/meter would be required, to overcome the resistance to flow as a result of the sieve plates. Thus, the Weatherly model, indicate that a tree 100 meters tall, would require a hydrostatic pressure of about 100 atm, in order to satisfy the pressure/hydrostatic model of phloem translocation. Crafts and Crisp have calculated the theoretical pressure required as about 9,2 X 10-" atm/meter, in order to drive a mass flow mechanism. When the cut stem pieces were increased in length to about 41cm, the effect of an increase in xylem pressure of 4 atm was greater, than was recorded in the 15 cm stem segments. Applications of respiratory inhibitors such as cyanide or dinitrophenol, at levels as low as Ix10-5 to 10-6 M, stopped exudation completely. Thus these early experiments, gave rise to profound results: - firstly, that the hydrostatic pressure of the xylem, influenced the rate at which exudation and presumably, the rate of translocation took place, secondly that the translocation system, was severely impeded by the application of metabolic inhibitors. Third, and most important, these pioneering results strongly supported the mass or hydro- static flow mechanism of phloem translocation.

 Virus movement

The translocation of viruses in plants has been studied for over 70 years, since the conclusion drawn by CW Bennett in 1927 that virus movement was correlated with food movement in plants. Much of the research that has been undertaken has emphasized the relationship between virus movement and phloem ontogeny.

It is important to realize that viruses differ in their tissue relations: some viruses are virulent in parenchymatous and vascular tissues, just as others are localized in the phloem. It is also important to bear in mind that plants differ in the rate at which they translocate organic assimilates - thus, there is no hard and fast rule which can be applied to the rate at which the viruses will be translocated though the plant. Tobacco mosaic virus moves slowly in parenchyma, but is translocated at the same rate as the organically assimilated foodstuffs, once it enters the sieve tubes of Nicotiana tabacum. Symplasmic movement of viruses, pesticides and tracer molecules occur in all plants. Accelerated in cell lumina by protoplasmic streaming, the rates of movement may reach rates of up to 5cm hr-1.

Transport rates

One further noteworthy generalization which has been substantiated with tracer work is the fact that  upon entering the phloem, exogenous substances will move rapidly from the source to sink. Mass rate of movement must depend upon the rate of entry into the phloem tissues (specifically the symplast), the rate of migration to the sieve tubes, the velocity of phloem translocation, the rate at which the substance is removed from the phloem by adjacent parenchymatic elements during transport and the metabolic activity of the sink. Thus, velocities of movement of up to 150 cm hr-1 are not unrealistic or improbable. 

Selected References

(Some of these are old, but are nonetheless, important background reading)

Aikman, D. P  Contractile proteins and hypotheses concerning their role in phloem transport.  Canad. J. Bot.  58 826-832. 1980. 

Ehlers, K., Knoblauch, M. and Van Bel, A.J.E.  Ultrastructural features of well-preserved and injured sieve elements: Minute clamps keep the phloem transport conduits free for mass flow.  Protoplasma  214 (1-2):80-92. 2000 

Eschrich, W.  Sealing systems in Phloem.  Encycl. plant Physiol. Transport in plants 1 phloem transport 39-56. 1975.

Evert, R. F and Mierzwa, R. J  Pathways of assimilate movement from mesophyll cells to sieve tubes in the Beta vulgaris leaf.  Phloem transport 419-432. 1986.

Fisher, D.B. and Cash Clark, C.E. Gradients in water potential and turgor pressure along the translocation pathway during grain filling in normally watered and water-stressed wheat plants.  Plant Physiology  123 (1):139-147.2000.

Lohaus, G., Heldt, H. and Osmond, C.  Infection with phloem limited Abutilon mosaic virus causes localized carbohydrate accumulation in leaves of Abutilon striatum: relationships to symptom development and effects on chlorophyll fluorescence quenching during photosynthetic induction.  Plant biol  2, 161-167. 2000.

MacRobbie, E. A C  Phloem translocation. facts and mechanisms: a comparative survey.  Biol. Review  46 429-481. 1971.

Oparka, K.J. and Cruz, S.S. The great escape: Phloem transport and unloading of macromolecules.  Annual Review of Plant Physiology and Plant Molecular Biology  51 323-347. 1040-2519. 2000.

Thaine, R. The protoplasmic theory of phloem transport.  J. Exp. Bot  45 470-484.1964.

Thompson, M.V. and Holbrook, N.M.  Application of a Single-solute Non-steady-state Phloem Model to the Study of Long-distance Assimilate Transport.  J. theor. Biol.  220 419-455. 2003.

van Bel, A.J.E. The Transport Phloem. Specifics of its functioning.  Progress in Botany  54, 134-150. 1993.

Weatherley P . Translocation in sieve tubes. Some thoughts on structure and mechanism. Physiol. Veg. 10, 731-742. 1972. 1964.

Weatherley P E . Solution flow in tubular semi-permeable membranes - short communication see

Weatherley P E, Johnson R P C . The form and function of the sieve tube: A problem in reconciliation. International review of Cytology 24, 149-192. 1968.

Weatherley P E, Peel A J, Hill G P . The physiology of the sieve tube. Preliminary experiments using aphid mouth parts. J. Exp. Botany 10, 1-16. 1959.

Zimmermann, M.H., Milburn, J.A. and Eschrich, W.  Transport in Plants I. Phloem transport.  Encyclopedia of Plant Physiology  1 (1):245-255. 1975.


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