Phloem transport m.sc c.u.

Phloem Translocation
 Brief anatomy of Phloem with special reference to Sieve Elements (SE) and
Companion Cells (CC)
 Pattern of Translocation: From Source to Sink
 Different functional sections of Phloem transport Pathways
i. Loading: Collection Phloem
ii. Dual Function (Leakage/Retrieval): Transport Phloem
iii. Unloading: Release Phloem
 Mechanism of Translocation: Pressure-Flow Model/Munch theory, Relay Model
 Loading Strategies
i. Apoplastic Loading
ii. Symplastic Loading/Polymer trapping Model
iii. Diffusion
 Unloading by Apoplastic/Symplastic Mechanism
 Phloem Sap Composition with special reference to Sucrose, P-Proteins, FT protein,
amino acids and small RNAs
 Sucrose transporters: Structures and types
 Signaling mechanism of Phloem Transport

Brief anatomy of Phloem with special reference to
Sieve Elements (SE) and Companion Cells (CC)
Differences with Xylem
Sieve elements (Sieve Cells and Sieve tube elements)
i. It looses nuclei and tonoplast during development.
ii. Absent of microfilaments, microtubules, Golgi bodies and ribosomes.
iii. Presence of modified mitochondria, plastids and SER.
iv. Non-lignified secondary thickened cell wall in some cases
v. Characteristic Sieve areas and large pores interconnect the conducting cells
vi. In angiosperms, Sieve areas differentiated to form Sieve Plates (having large pores
than other sieve areas, generally found on the end wall)
vii. Individual cells of the sieve elements stacked end to end to form Sieve tube.
viii. In angiosperm, pores on the sieve tubes are considered as open channels while in
Gymnosperm it is found to be blocked with SER.

Companion Cells
• Companion cells are parenchymal cells
• They possess the full complement of cytoplasm and organelles, numerous
plasmodesmata.
• Derived from same progenitor cell as the sieve-tube member
• Live and die together
Functions:
• Role in transport of photosynthesis products from producing cells in mature
leaves to sieve plates of the small vein of the leaf
• Synthesis of the various proteins used in the phloem
• Contain numerous mitochondria for cellular respiration to provide the cellular
energy (as ATP) required for active transport

Types of Companion Cells
• Ordinary Companion cells:
– Chloroplasts with well developed thylakoids, smooth inner cell wall,
relatively few plasmodesmata.
• Connected only to it’s own sieve plate
• Transfer cells:
– Well developed thylakoids
– Have fingerlike cell wall in growths –increase surface area of plasma
membrane for better solute transfer.
Both of these types are specialized for taking up solutes from
apoplast or cell wall space

Types of Companion Cells
• Intermediary cells:
• Appear well suited for taking up solutes via cytoplasmic connections
• Have many plasmodesmata connects to surrounding cells
• Most characteristic feature
• Contain many small vacuoles
• Lack starch grains in chloroplast
• Poorly developed thylakoids
Function in symplastic transport of sugars from mesophyll cells to
sieve elements where no apoplast pathway exists

 Brief anatomy of Phloem with special reference to Sieve Elements (SE) and Companion
Cells (CC)
 Pattern of Translocation: From Source to Sink
 Different functional sections of Phloem transport Pathways
i. Loading: Collection Phloem
ii. Dual Function (Leakage/Retrieval): Transport Phloem
iii. Unloading: Release Phloem
 Mechanism of Translocation: Pressure-Flow Model/Munch theory, Relay Model
 Loading Strategies
i. Apoplastic Loading
ii. Symplastic Loading/Polymer trapping Model
iii. Diffusion
 Unloading by Apoplastic/Symplastic Mechanism
 Phloem Sap Composition with special reference to Sucrose, P-Proteins, FT protein,
amino acids and small RNAs
 Sucrose transporters: Structures and types
 Signaling mechanism of Phloem Transport
Phloem Translocation

Pattern of Translocation: From Source to Sink
Photosynthesis provides a
sugar source
New growth is a
sugar sink
Translocation
Direction of transport through phloem is
determined by relative locations of areas of
supply, sources and areas where utilization of
photosynthate takes place, sinks.
Source: any transporting organ capable
of mobilizing organic compounds or
producing photosynthate in excess of its own
needs, e.g., mature leaf, storage organ during
exporting phase of development.
Sink: non photosynthetic organs and
organs that do not produce enough
photoassimilate to meet their own
requiements, e.g., roots, tubers, develpoping
fruits, immature leaves.

Source to Sink: A pattern is followed
Sieve elements of Sources
Sieve Elements of Connecting Pathway
Sieve Elements of Sinks
Collection Phloem
Transport Phloem
Release Phloem
• Although the overall pattern of transport are generally from source to sink
• Not all sources supply all sinks in a plant
• Certain sources preferentially supply specific sinks
Examples:
Beta maritima (wild beet) root is a sink during the first growing season. In the
second season the root becomes a source, sugars are mobilized and used to
produce a new shoot.
In contrast, in cultivated sugar beets roots are sinks during all phases of
development.

Factors effecting the Pattern
• Proximity: – of source to sink is a significant factor.
– Upper mature leaves usually provide photosynthesis products to
growing shoot tip and young, immature leaves
– Lower leaves supply predominantly to the root system
– Intermediate leaves export in both directions
• Development: – Importance of various sinks may shift during plant
development
– Roots and shoots major sinks during vegetative growth
– But fruits become dominant sinks during reproductive
development

Factors effecting the Pattern
• Vascular connections: –Source leaves preferentially supply sinks with direct
vascular connections
– A given leaf is connected via vascular system to leaves above and below it
on the stem
• Modifications of translocation pathways: - Interference with a translocation
pathway by mechanical wounding (or pruning)
– vascular interconnections can provide alternate pathways for phloem
transport
 The flow of water in plants is almost always from roots to
leaves.
 Translocation of sucrose can be in any direction – depending on
source and sink location and strength.

How can you identify the pattern of phloem translocation?
Girdling a tree, i.e., removing a complete ring
of bark and cambium around a tree, has no
immediate effect on water transport, but sugar
accumulates above the girdle and tissue swells
while tissue below the girdle dies.
Girdling experiments
*Girdling is sometimes used to enhance fruit production..
Radio active tracer experiments
Application of 14CO2 to a photosynthesizing leaf, or application
of 14C-sucrose, then visualization of the path of the radioactive
tracer through radiographing cross sections of the plant’s stem
indicates that photosynthate moves through phloem sieve
elements.

Different functional sections of Phloem transport Pathways
Loading/Collection: In the Collection phloem assimilates are loaded into sieve
element-companion cell complex (SECCC) of minor veins after being
produced in the mesophyll
Transport: Assimilates are transported towards sinks via transport phloem.
Transport phloem is located in the major veins , petioles, branches, stem and roots
Terminal Sink
(Shoot, root)
Lateral Sink
(Cambium)
Continuous leakage and
retrieval of solutes
Unloading /Release: The main function of release pholem in the sinks is to
unload the assimilates from SECCC into growing or storage cells
Balance between release and retrieval varies with the requirements of the plant.

 The solute concentration, and implicitly the
turgor, are controlled by release/retrieval
mechanisms in the sieve element–companion
cell complexes (SECCCs). Differential
release/retrieval balances control the
influx/efflux of sugars (violet arrows) and water
(blue arrows) in the various phloem zones.
 The gradual loss of solutes and commensurate
amounts of water towards the sink, where
massive delivery of water and solutes takes
place, has been ascribed to the relative size
reduction of CCs along the source to sink path,
which may explain a decreasing retrieval
capacity of the SECCCs in the direction of the
sink.(Schepper et al., 2013)

According to Pressure flow (Munch theory) loading and unloading only occurs in source
and sink respectively. However, loading and unloading mechanism is also operated in
transport phloem which is known as Leakage retrieval mechanism (van Bel, 2003;
Thorpe et al., 2005)
How?
Sieve tubes are considered as permeable
Pressure gradient is negligible during transport along the phloem which regulates
the solute exchange between the SECCC and surrounding cells
Without a nervous system (Decentralized plant body) can only sense and modify
their own turgor not the turgor gradient
Thompson Hypothesis
(Thompson, 2006)
Supporting Reason

Mechanism of Translocation: Pressure-Flow
Model/Munch theory, Relay Model
Translocation is thought to move at 1
meter per hour
– Diffusion too slow for this speed
• The flow is driven by an osmotically
generated pressure gradient between the
source and the sink.
• Sugars (red dots) is actively loaded into
SECCC (Source)
• Called phloem loading
• Sugars are unloaded into sink
• Called phloem unloading

• yw = ys + yp + yg
• In source tissue, accumulation of Sugars
– Makes low (-ve) solute potential
– Causes a steep drop in water potential
– In response to this new water potential gradient,
water enters sieve elements from xylem
• Thus phloem turgor pressure increases
3 mechanisms exist to generate high
concentrations of sugars in Source
i. Photosynthetic metabolism in
mesophyll (C3 metabolic cycle)
ii. Conversion of photoassimilate to
transport sugars in intermediary cells
iii. Active membrane transport
Pressure-Flow Model/Munch
Theory

In sink tissue, phloem unloading leads to lower sugar conc.
Makes a higher (+ve) solute potential
Water potential increases
Water leaves phloem and enters sink sieve elements
and xylem
Thus phloem turgor pressure decreases
• So, the translocation pathway has cross walls
– Allow water to move from xylem to phloem and back
again, if absent- pressure difference from source to
sink would quickly equilibrate
• Water is moving in the phloem by Bulk Flow
– No membranes are crossed from one sieve tube to
another and solutes are moving at the same rate as the
water
Water movement is driven by pressure gradient and NOT water potential gradient
Pressure-Flow
Model/Munch Theory

Short comings of Munch Theory
 No true bidirectional transport
 No great expenditures of energy are required in order to drive the translocation
Therefore, lower supply of energy (ATP) during low temp. anoxia, metabolic
stress should not effect translocation
 The lumen of sieve tube and the sieve plate pores must be largely unobstructed.
If P-proteins/callose blocks the pores, resistance develops.
 Pressure difference must be large enough to overcome the resistance pathway
and to maintain the velocities.
FPc =
Psource -Psink
RPc
Pressure gradient should be larger in trees (long distance) than herbs (short distance)
In large trees , RPc also becomes very large and thus a point might be reached where
the pressure gradient required to drive the Munch Flow exceeds the turgor pressure
that can be generated…So Munch theory for herbs or trees?????

Support for Munch Theory
 No bidirectional movement in a single SE
 Transport in two directions is found in SEs of different VB (vascular
bundles), adjacent SEs of same VB like petiole where exporting and
importing of sugar take place.
By Radiotracers to two source leaves (one above other), Each leaf receives
one of the tracers and a point between the two sources is monitored.
 Solutes and water move at the same velocity
By carbon labeled solutes or NMR the water velocity is indicated …similar
to velocity of solute transport

Support for Munch Theory
 Lack of energy requirement in the pathway of herbaceous plant
Treatment with metabolic inhibitor (cyanide) impedes the translocation in the petiole of
source leaf (in bean). Interestingly examination of treated tissue by Electron
microscopy reveals the blockage of the sieve plate pores by cellular debris
 Presence of open sieve plate pores
Rapid freezing, fixation and Confocal scanning electron microscopy
provides the direct evidence of translocation through SE. Open sieve plate pores
have been found in many species..
Does P-protein/SEOR protein/any protein block the lumen???

Relay Model
Some studies suggested that in some large trees the sieve tubes are
shorter than Plant axial length. The proposed translocation
pathway is composed of a series of shorter overlapping sieve tubes
with apoplastic loading steps between them. Lang (1979) called
those intervening loading steps “Relays”.
Solutes are energetically transported from one unit to next unit
provides a pressure boost…….
Osmotically generated pressure flow is the main thrust for the
translocation like Munch theory. In addition speed and directions
are also controlled during relay transport.
Leakage –Retrieval mechanism is considered as dynamic Relay
model (van Bel 2003, 2005)

 Phloem sap is rich in organic molecules and ions…needs a
elaborated set of transporters unless the composition of sap
changes at each step.
 Anatomical studies of Phaseolus vulgaris do not support the
hypothesis
 Resistance in transport phloem is proportional to the plant length,
the resistances in the Collection and Release phloem are inversely
proportional to the source (leaf length) and sink (root length).
Altered relationship of resistance for Relay Model.
Difficulties of Relay Model
R3α Ll x S l (For herbs and trees) [R= sieve tube radius, Ll= leaf length, Sl= Stem length]

Differences of phloem transport between herbs and trees

Phloem Loading
• In a process called sieve element
loading, sugars are transported into
the sieve elements and companion
cells
• Sugars become more concentrated in
sieve elements and companion cells
than in mesophyll cells
• Once in the sieve element /companion
cell complex, sugars are transported
away from the source tissue – called
export
• There are 3 possible ways for the
sucrose to enter the phloem sieve
element cells
– Active apoplastic loading
– Active symplastic polymer
trapping
– Passive symplastic diffusion
Translocation to the sink tissue is
called long distance transport

Apoplastic loading
• Active transport against it’s chemical
potential gradient
• Efflux into the apoplast is highly
localized, probably into the walls of
phloem parenchyma cells.
• Mesophyll cell SECCC (Transfer
type companion cell)
• Sucrose uptake in the apoplastic pathway
requires metabolic energy…Osmotic
potential
• Osmotic potential in mesophyll cell
should be higher as compared to
SECCC.
• Involves a sucrose-H+ symporter
The energy dissipated by protons moving back into
the cell is coupled to the uptake of sucrose
Treating source tissues with respiratory
inhibitor decreases ATP concentration and
sugar export
Experimental evidence
Mainly in
herbs

Active symplastic loading: Polymer trapping model
• Depends on plant species
– Depends on species that transport sugars other than sucrose
• Requires the presence of open plasmodesmata between different cells in the
pathway
• Plant species with intermediary companion cells.
• Sucrose, synthesized in mesophyll, diffuses into intermediary cells, more
concentrations of sucrose in mesophyll cell than intermediary cell
• Raffinose and stachyose is synthesized from sucrose and galactinol. Due to
larger size, can NOT diffuse back into the bundle sheath cell.
• Raffinose and sucrose are able to diffuse into sieve element

Why polymer trapping is called active symplastic
loading?
Although it does not involve the active transport in the formal
sense of moving ions and molecules across a membrane. It is
thermodynamically active since energy is used to create
concentration difference between the mesophyll cells and the
SECCCs.
 Found mainly in herbs and trees

Passive symplastic Diffusion
• A large number of woody trees load assimilates passively by maintaining high
sucrose conditions, and in some cases sugar alcohols in the mesophyll cells.
• It requires no energy
• Sugar levels are higher in mesophyll cells than in phloem
• Solutes diffuse from ordinary companion cells through plasmodesmata.
3 critical factors of phloem loading:
i. Turgor pressure: regulates symplastic loading, H+ATPase activity which
influences the PMF that drives the sucrose carriers…regulate apoplastic loading
ii. Sucrose level: Controls the diffusion and activity of sucrose carriers
-High turgor and/or sucrose levels in SECCC will decrease phloem loading
iii. Presence of phytohormones: cytokinins, ABA and GA also regulates phloem
loading

Phloem Unloading : Import into sinks
• Three steps
• (1) Sieve element unloading:
– Transported sugars leave the sieve elements of sink tissue
• (2) Short distance transport:
– After sieve element unloading, sugars transported to cells in the sink by means
of a short distance pathway
• (3) storage and metabolism:
– Sugars are stored or metabolized in sink cells
• Also can occur by symplastic or apoplatic pathways
• Varies greatly from growing vegetative organs (root tips and young leaves) to
storage tissue (roots and stems) to reproductive organs
Symplastic Phloem Loading
• Appears to be a completely symplastic pathway in young dicot leaves
• Again, moves through open plasmodesmata

• Apoplastic: three types
• 1 [B] One step, transport from the sieve element-companion cell complex to
successive sink cells, occurs in the apoplast.
• Once sugars are taken back into the symplast of adjoining cells transport is
symplastic
Apoplastic phloem loading

(2) [A] involves an apoplastic step close to the sieve element
companion cell.
(2) [B] involves an apoplastic step which is farther removed from the
SECCs (In seeds sucrose transported maternal tissue to embryo)
Both involve movement through the plant cell wall

Phloem sap proteins
P proteins:
• Several forms like tubular, granular, fibrillar, crystalline in mature cell and discrete bodies as
p-protein bodies in the cytosol
• Well known class of SE proteins involved in the plugging of sieve pores are PP1 and PP2
High molecular weight polymers
Close the sieve pores
Under oxidative conditions, PP1 monomers and PP2
dimers are covalently cross-linked via disulphide bonds
With the help of the callose (b-1,3-glucan),
accumulates on sieve plates after different stress
treatments to prevent assimilate loss from cut SEs
Rapid, Reversible
Long term, irreversible
wound/injury, biotic stress Calcium ions

• In Monocot:
P-protein released as crystals from ruptured plastids , SE organelles appeared to
be anchored to each other or to SE plasma membrane by minute protein
“clamps”
• In Legume:
Large crystalloid P –protein, rapidly disperse and block the Sieve tube in
response to stress with the help of calcium ions….known as Forisomes (by SE
occlusion protein group member)
• Lectin (sugar binding proteins) and protease inhibitors:
PP2 like Lectin protein in Arabidopsis,
Trypsin, chymotrypsin, serine, and aspartic protease inhibitors and cysteine
protease inhibitors in Ricinus
• Other defense related proteins

FT-protein
Mobile protein moves between the SECC and surrounding tissues of sources and
sinks
Floral stimulus moves from companion cells of source leaf to apex via
plasmodesmata and induces flowering at the apex.
Movement through plasmodesmata can be either passive, selective and regulated
Selective transport through plasmodesmata
•Molecule moves passively: size must be smaller than the size exclusion limit
(SEL) of the plasmodesmata
•Molecule moves through a selective pathway: size larger than SEL proteins
directly interact with components at or within the plasmodesmata..with the
help of chaperones thay can move through a selective pathway..

RNAs as mobile element
Endogenous mRNA, pathogenic RNAs, small RNAs associated with gene silencing can
travel through phloem as ribonucleoprotein complexes.
Evidence of RNA transport: Unloading of mRNAs in sink tissues
•GAI (Gibberelic acid insensitive), mRNAs for Gibberelic acid localized to SECC of
pumpkin and phloem sap. mRNA for mutant regulator (transgenic plants) was localized to
SEs, able to be transported across graft unions into WT Scions, unloaded to apical tissues
Motifs in coding region and in the untranslated region of GAI RNA and
BEL5 are mostly responsible for phloem transport
BEL5, another mRNA of in potato formed in the leaves moves through the phloem to
the stolon (site of tuber production)
Provide a signal for enhanced tuber production

Sugar transport
•Nonreducing sugars are the major compounds translocated in the phloem because
of their less reactivity than the reducing sugars
•Hexoses cannot be tolerated in the phloem, in which very high sugar levels are
maintained. They are sequestered in the vacuoles and have no direct connection
with the phloem
Raffinose: Sucrose + galactose
Stachysoe: Sucrose+ 2 galactose
Verbaose: Sucrose + 3galactose
Sugar alcohols: mannitol and sorbitol
Allocation of sugar moieties is very
important which includes storage,
utilization and transport
Allocation
Synthesis of storage compounds: Starch within the chloroplast
Metabolic utilization: fixed carbon can be utilized by photosynthesizing cells
Synthesis of transport compounds: fixed carbon can be incorporated into
transport sugars for export

Various sinks partition transport sugars: Determines the patterns of
growth, must be balanced between shoot and root growth
Sucrose/H+ symporters have been
localised to the plastid (AtSUT4),
the tonoplast membrane (AtSUT4,
HvSUT2) and the plasma
membrane Energy-independent
sucrose transport mediated by
facilitators has been reported on
the tonoplast.
Membrane in sink storage cells
and function as effluxers on the
plasma membrane of legume seed
coats and root tips.
There is also a strong biochemical
evidence of a proton coupled
antiport mechanism operating on
the plasma membrane of bean
seed coats
Various Sucrose Transporters

Phloem transport m.sc c.u.

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Phloem transport m.sc c.u.