# 9. Phloem
transport mechanisms - some
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:
is important to
The loading process:
can follow either a passive pathway,
the first instance,
there may be no energy or thermodynamic demands
placed upon the system.
the second, ATP
& NADPH would
be needed directly to drive co-transport across membranes.
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.
of sucrose to starch reduces 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
These can beplaced in one of two categories.
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
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.
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.
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 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
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),
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
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
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
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
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.
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.
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.
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
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.
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
(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.