After reading this article you will learn about: 1. Phloem Sap Composition 2. Movement in Phloem Sap.
Phloem Sap Composition:
The major phloem sap components are carbohydrates. Analyses of the phloem exudates from various plants have shown that sucrose is the major transportable form of carbohydrate. In some species of Cucurbitaceous, in addition to sucrose, certain oligosaccharides like raffinose, stachyose, and verbascose have also been found in the phloem sap composition.
Again in some cases sugar alcohols mannitol and sorbitol or dulcitol have been found in the phloem exudates. Generally, the seaweeds produce large amounts of mannitol. Phloem exudate rarely contains hexoses even though glucose and fructose are commonly present in phloem tissue. They are the products of sucrose hydrolysis and are distributed in non-conducting cells to enter directly into the metabolism.
The non-reducing sugar, sucrose, on the other hand, is a more stable compound and suitable for long-distance transport. For that reason, it has been found that the ratios of labeled hexoses to labeled sucrose decreased as the distance from the leaf assimilating 14CO2 increased.
Exudate analyses for nitrogenous compounds of willow stems have detected the presence of glutamic acid, aspartic acid, threonine, serine, leucine, alanine, valine, phenylalanine, asparagine, glutamine, etc. Phloem exudate also contains high levels of proteins, particularly P- protein and several enzymes of carbohydrate and nitrogen metabolism.
The enzymes of glycolysis, TCA cycle, pentose phosphate pathway, transamination, peroxidase, polyphenol oxidase, etc., have been found to be present in the sieve elements.
The protein profile also includes some proteins associated with basic cellular functions like protein kinases (protein phosphorylation), thioredoxin (disulfide reduction), ubiquitin (protein turnover), and chaperones (protein folding).
All these proteins are called sieve tube exudate proteins (STEPs).
These are grouped as:
(1) Enzymes related to carbohydrate metabolism,
(2) Structural proteins (“P-proteins”)
(3) “Maintenance” proteins.
The structural proteins appear as either crystalline or amorphous accumulations (“P-protein bodies”) during sieve element differentiation. Though common, these structures are absent in some species, including many monocots.
The amorphous, fibrillar forms of this protein appear to be in equilibrium with soluble monomeric proteins, which may be fairly abundant in phloem exudates. Because of their abundance in cucurbit exudates, these forms have been characterized fairly extensively.
Several forms are present in phloem exudates from a given species, and exudates collected from different organs on a plant demonstrate similar protein patterns. However, both gel patterns and antibody cross-reactivity data suggest the existence of significant differences among species.
A particularly interesting aspect is the hem-agglutinating (lectin) activity of some P-proteins, which has provided another useful diagnostic tool for probing taxonomic relationships.
Its biological or physiological significance, however, is unclear. Recent efforts at characterizing STEPs have focused on their possible maintenance role in SE/ CC (sieve element/companion cell) interactions. Identified functions in this category include glutaredoxin, cystatin, ubiquitin, and chaperones, thioredoxin and protein kinase activity.
The phloem sap also contains high levels of K+ and Mg2 +. Among the anions Cl– and PO43-are very common. Traces of zinc, manganese, copper, iron and molybdenum are also present. Probably, due to its high K+ content the phloem exudate is slightly alkaline.
Growth substances like indole acetic acid, gibberellic acid and abscisic acid have been detected in phloem sap. In addition, organic acids, particularly malic acid, nucleic acids, ATP, vitamins, etc., have also been detected in phloem sap. So, phloem is an important path for the translocation of various materials.
As a general rule the sieve-tube sap is the most concentrated solution to be found in any space of the plant body and has the most negative osmotic potential (Ψs).
Movement in Phloem Sap:
Photosynthetic are trans-located from the site of production (source) to the site of utilization or storage (sink). The flow rate is proportional to the gradient of pressures in the phloem. Different substances are trans-located at different rates.
Translocation results in the transport of dry matter away from sources or into sinks. It is expressed in terms of cross-sectional area of phloem. The quantity of dry matter transferred per unit time per cm2 of phloem is termed the specific mass transfer (SMT, gh-1 cm-2 ph).
This measure was used by Mason and Maskell. Concentration gradient of the phloem sap from source to sink is the important factor for phloem transport. From the origin of translocation the concentration declines along the pathway towards the sinks, so that exudates taken from different heights of a tree show a progressive decline in concentration downwards.
The high solute concentration of the sieve-tube sap contained within the plasmalemma generates a large osmotic potential, and hence a large turgor pressure in the sieve tubes. Dixon calculated the speed of the sap from the equation.
Mass transfer per unit area = Concentration of sap x Speed.
If there exists a solute concentration gradient along the sieve tubes there will also exist a turgor pressure gradient, which is one possible force to drive the flow. Mason and Maskell showed that the SMT was proportional to sucrose concentration in the phloem and pointed out that this relation is the same as Fick’s law of diffusion.
Direction of Movement in Phloem Sap:
Phloem translocation is not exclusively in either an upward or a downward direction. It is not defined with respect to gravity. Generally, photosynthesis occurs in the leaves and the photosynthates are trans-located to the sinks. From the thousands of experiments regarding the direction of phloem sap movement a few simple rules have become known.
The rules are as follows:
1. The mature leaves never behave as sinks. The growing leaves before attaining about half the maximum size, act as importers or sinks, i.e., sap movement takes place into them. Then they switch over to the exporter stage and remain the same for the rest of their lives.
2. From the upper leaves most of the solutes move upwardly into the stem apex and from the lower leaves to the roots by downward translocation. Leaves in the middle portion feed both through bidirectional movement.
3. The pattern of movement may be changed by the removal of sources or sinks. If the apical leaves are removed, the lower leaves then export most of their output apically.
4. From autoradiographs of stem sections it is revealed that the solutes travel in straight lines along the phloem with very little sideways spread.
5. Active sinks are fed by the nearest source.
Sources include any exporting organ, typically a mature leaf, capable of producing surplus photosynthates. Storage root of the biennials may also be the sources under some circumstances. It behaves as a sink during the growing season of the first year when it stores food received from the source leaves.
During the following growing season the same root becomes a source. The sugars are remobilized and utilized to produce a new shoot. Sinks include any non-photosynthetic organs that do not produce their own food.
(a) Bidirectional Movement:
So, it is evident that the trans-locates move within the phloem in bidirectional pattern — that is, in opposite directions simultaneously. To feed the roots, the photosynthates move downwards, or they may move upwards towards growing points, where flowers, young leaves, or fruits are developing. The tap roots, tubers, bulbs, etc., are fed through downward translocation.
In the growing seedlings the stored foods in the cotyledons, tubers, bulbs, etc., are trans-located in an upward direction.
The translocation of materials out of the aging leaves and into young leaves is an upward movement. Many leaf traces do not join the stem vasculature at the node point, but at the node below or even the farther one. Hence, photosynthates from a leaf travel down the stem one or two nodes before entering the stem vasculature.
It has been found through tracer techniques that photosynthates move in both directions in the stem simultaneously. Whether the movement in different directions takes place in different phloem ducts or in the same duct simultaneously is still unknown. Biddulph and Cory (1957), however, have shown that bidirectional movement in bean plants takes place in separate phloem bundles.
Wareing and Patrick (1975) introduced the idea of sink-strength based on the observation that at a particular stage of development some sinks dominate over others, attracting the major portion of trans-locates from all sources. The sink-strength depends on the size and activity of the sink.
Sink strength = Sink size x Sink activity
Sink activity is the rate of uptake of photosynthates per unit weight of sink tissue, and sink size is the total weight of the sink tissue.
(b) Lateral Movement:
According to the rule 4 of the phloem transport, trans-locates travel in straight lines along a narrow band of phloem. Sugars moving out of a leaf into the main translocation stream will move both up and down the stem in the same vertical line.
Labelled translocate from one leaf, entering a single vascular strand in the stem, may be confined to that strand for a long distance. Very little lateral movement takes place. One-sided defoliation of a plant results in asymmetrical growth and the defoliated side shows reduced growth.
Joy (1964) studied translocation patterns in sugar beet. 14CO2 was fed to a leaf for four hours. After one week he found labeled trans-locates only in leaves above on the same vertical line, or in the roots just below the supplying leaf, indicating the absence of lateral movement.
But when he removed all fully expanded leaves except the young immature leaves from one side of the plant and then fed 14CO2 to a mature leaf on the opposite side, labeled trans-locates were detected from the young intact leaves on the defoliated side. It indicated lateral movement in tangential direction. The young leaves acted as strong nutrient-attractive sites.
i. Source-Sink Relationship:
Photosynthetic rate and SMT very often depend on source-sink ratio. They increase over several days when sink demand increases and decreases when sink demand decreases.
When all but one of the source leaves are removed for a longer period, many changes occur in the source leaf including increase in photosynthetic rate, sucrose concentration, phloem transport, orthophosphate concentration, and decrease in starch accumulation. Photosynthesis, on the other hand, is strongly inhibited under conditions of reduced sink demand.
Thus, there appears a metabolic adjustment in the source in response to the altered conditions.
When sink demand decreases high starch levels in the source disrupts the chloroplasts interfering with CO2 diffusion and light absorption. Under such circumstances the phosphate availability in the chloroplasts is lowered as its exchange with triose phosphates decreases leading to decline in ATP synthesis and CO2 fixation.
In low sink demand high sugar levels in the source cells decrease the transcription rate and expression of genes for many photosynthetic enzymes (Koch 1996). During metabolic adjustment under high sink demand de-repression of the repressed genes occurs.
The long distance signals coordinate the activities of sources and sinks. Signals between them might be physical or chemical. The physical signal is turgor pressure, whereas the chemical signals are plant hormones and carbohydrates.
ii. Phloem Loading:
In the leaf phloem sucrose concentration remains very high and, therefore, osmotic potentiality is also very negative. It has been found that mesophyll cells have an osmotic potential of about -1.3 to – 1.8 MPa, whereas leaf sieve elements have an osmotic potential of about – 2.0 to – 3.0 MPa.
So, from the source ceils (mesophyll cells), sugar is somehow transported and concentrated in the leaf phloem cells. The process in which sugars are raised to high concentrations in leaf sieve elements close to the source cells is called phloem loading. This origin of sucrose gradient and the pressure gradient has been an active area of research in recent years. Most of the loading occurs in the minor leaf veins.
In leaves with reticulate venation the smallest veins are the first to show accumulation of 14C-assimilate above the level in the surrounding mesophyll. In parallel venation the very small transverse connecting veins in between pairs of large veins, are loaded first, and longitudinal translocation takes place through the large veins.
The trans-locates have to pass through no cells at all or few cells to reach minor leaf veins. The distance between mesophyll cells and sieve tubes is not more than 100 µm. There are two possible routes for the sugar traffic for this short distance, an apoplastic (cell walls outside the protoplast) and a symplastic (cell to cell through plasmodesmata) route.
There is considerable evidence that sucrose is actively transported or secreted out of mesophyll cells into the apoplast of the minute veins from where the sugar is absorbed actively into the companion cells. From the companion cells the sugars then pass through the symplast into the sieve elements.
Secretion of sucrose from the mesophyll cells into apoplast and subsequent loading of companion cells and then sieve tubes are active processes. Several recent studies reviewed by Giaquinta suggest that active loading of sucrose into the phloem occurs by a cotransport system.
It follows the Mitchell’s scheme for translation of the energy of ATP by vectorial ATPases in membranes coupled to the transport of protons across the membrane.
The energy for sucrose accumulation against a concentration gradient is generated by the extrusion of protons (H +) from the space at the expense of ATP, and then as the protons return across the membrane, cotransport of sucrose takes place by a membrane-bound sucrose-specific ‘symport’ carrier system.
As a result, sucrose molecules accumulate within the space. Such sucrose/proton cotransport pumps have been found in the cotyledons of Ricinus, which transport sucrose from the endosperm during germination.
The transport is stimulated by fusicoccin and inhibited by dinitrophenol (DNP), valinomycin, p-chloromercuribenzene sulphonic acid (PCMBS), etc., possibly by affecting the membrane-bound sucrose carrier.
When sugar-beet leaves were allowed to assimilate 14CO2 PCMBS. markedly inhibited the translocation of assimilates through the phloem, suggesting strongly that sucrose produced in photosynthesis must enter the apoplast before being loaded into the phloem. From the apoplast sucrose is actively loaded into companion cells and then the sieve elements.
These electro genic proton pumps do not only push H+ to one side of a membrane, but also exchange it for K+ moving in the opposite direction to maintain the electrical neutrality (Fig. 6.6). For that reason, K+ is universally present in sieve tube exudates.
There are many evidences for sucrose/proton cotransport:
(i) The contents of sieve tubes are known to have a relatively low proton concentration (pH 7.5 to 8.5) and high potassium concentration.
(ii) The apoplast at the loading sites is quite acidic compared to the phloem sap and if the pH of the apoplast of a leaf is increased, sucrose uptake is inhibited.
(iii) PCMBS inhibits sucrose uptake in sugar beet leaves with the concomitant inhibition of proton secretion by the phloem minor veins.
Young leaves usually act as sinks and attract nutrients from the mature leaves. But just when the companion cells of the minor veins of immature leaves develop phloem loading capacity they change over from this import mode to an export mode of transport and become sources instead of sinks.
Among the sucrose carriers SUT- 1 (42 kDa) has been isolated from the cell membrane of sugar beet . pS21 (55 kDa) from spinach. A sucrose transporter SUC1 (54.9 kDa) was isolated from transgenic yeast, and another SUC2 (54.5 kDa) was isolated from Arabidopsis thaliana and Plantago major.
These carriers are highly specific to sucrose and the amino acid sequences of these sucrose transporters obtained from different sources have a high degree of homology. High levels of both SUC1 and SUC2 gene expressions were observed in mature leaves, particularly in the phloem, but low expression in sink tissues where the monosaccharide transporters dominate.
The sucrose transporter SUC2 is expressed only in the companion cells of the phloem and not in the sieve elements. This suggests the major role of companion cells in phloem loading. Sucrose accumulates first in the companion cells and then diffuses into the sieve elements through many plasmodesmatal connections.
The driving force for the uptake of sucrose into companion cells is provided by the pmf generated by the plasma membrane H + -ATPase AHA3 isoform in Arabidopsis. The sucrose transporter SUT1 in Solanaceae is present in sieve elements, not in companion cells. SUT1 mRNA is found in the mature sieve elements devoid of nuclei in greater amounts than in companion cells.
It suggests that SUT1 mRNA is transported along with sucrose through plasmodesmata from the companion cells to the sieve elements, where it is translated. The sucrose transporters are the members of the major facilitator super family. They are hydrophobic and contain single subunit with 40-50 kDa molecular mass. They have 12 trans-membrane domains, with hydrophilic loops.
Depending on the species, phloem loading in leaves may occur by trans-membrane movement (apoplastic loading) or via plasmodesmata (symplastic loading). In some species, sieve element/ companion cell (SE/CC) complex remains symplastically isolated. High osmotic concentration of the phloem exudates is observed in comparison to that of mesophyll cells.
These findings suggest that sucrose is absorbed directly from the apoplast against the concentration gradient through active absorption process. Most crop species translocate sucrose and are apoplastic phloem loaders. The cucurbits translocate raffinose series of oligosaccharides (RSOs).
Their minor veins are incapable of absorbing RSOs from the apoplast. These plants and many others of different families exhibit high plasmodesmal frequencies between the minor vein companion cells and adjacent bundle sheath cells.
So, depending on minor vein configurations there are two groups of plants – Type-1 and Type-2 species. The type-1 species with “open” minor vein configuration are symplastic loaders and type-2 species with “closed” minor vein configuration are apoplastic loaders. Type-1 species exhibit extensive plasmodesmal connections, whereas in type-2 the SE/CC complex appears to be symplastically isolated.
They absorb solutes from the apoplast by membrane transport. Sucrose moves symplastically from the mesophyll to the minor veins, where it is released from vascular parenchyma cells to apoplast for subsequent absorption into the SE/CC complex by H + -sucrose cotransport.
In fact the type of phloem loading is correlated with taxonomic groupings (families) and climate. In general, extensive plasmodesmatal connections between phloem and surrounding cells are found in plants growing in tropical and subtropical regions, whereas plants growing in temperate and arid climates have few plasmodesmata at this interface.
The symplasmic movement to the minor veins is supported by the observed high frequencies of plasmodesmata linking the cell types as well as by the results of dye coupling experiments. Symplastic continuity from the mesophyll to the SE/CC complex allows assimilates to move directly into the translocation stream.
The mechanism of its operation is unclear as there is no known process for active solute transport through plasmodesmata against a concentration gradient. In vascular plants the basic plasmodesmal structure is a tube of plasma membrane surrounding a strand of modified endoplasmic reticulum, with particulate material between them.
The greatest mechanistic challenge appears in RSO trans-locators where movement into SE/CC complex occurs against a concentration gradient. For these species, a “polymer trapping concept” has been proposed to account for the major characteristics of phloem loading.
In this concept, sucrose is synthesized in the mesophyll and moves via plasmodesmata to companion cells where sucrose is used to synthesize the larger RSO molecules. It is supported by the fact that all the enzymes required to synthesize stachyose and raffinose are present in high concentrations in the companion cells than in mesophyll cells.
iii. Phloem Unloading:
The phloem unloading can be defined as the movement of photosynthates from the sieve elements and their distribution to the sink cells that store or metabolize them. The unloading events in sink tissues are simply the reverse of the events in sources. Translocation of solutes into sinks, such as roots, tubers, and floral structures, is termed import.
The following steps are involved in the import of sugars into sink cells:
1. Sieve element unloading. By this sieve element unloading process the imported sugars come out of the sieve elements of sink tissues.
2. Short-distance transport. After sieve element unloading, the sugars are transported to the sink cells through a short-distance transport pathway, also called post-sieve element transport.
3. Storage and metabolism. In the final step, sugars are stored or metabolized inside the sink cells.
The above three transport steps together constitute phloem unloading. It can occur via symplastic or apoplastic pathways.
Sinks may be of different types like growing roots and leaves, storage tissues, reproductive structures such as fruits and seeds. Due to the great variations of sink’s structure and function, there is no single scheme of phloem unloading. The sugars may be transported entirely through the symplast, or through the apoplast in some regions.
The unloading pathway in some young dicot leaves, such as sugar beet and tobacco is completely symplastic. Sufficient plasmodesmata exist in these pathways to support symplastic unloading. It is also supported by the fact that the efflux rates typically exceed membrane transport rates by an order of magnitude or more.
The use of fluorescent tracers such as carboxyfluorescein (CF) has allowed direct observation of sieve element unloading in several sinks. CF moves through phloem with very little loss, and on arriving in a sink it is unloaded in the same pattern as endogenous solutes. This confinement of CF to the symplasm clearly indicates that unloading from the SE/CC complex occurs via plasmodesmata.
For the sieve tube unloading steps a large differential pressure exists between the SE/CC complex and the surrounding cells of the sink originated by the high hydraulic resistance of plasmodesmata and the universally high concentration of the SE/CC contents.
This differential pressure in wheat grains is approximately 1.0 MPa and in barley root tips it is about 0.7 MPa.
The phloem is viewed as a high-pressure manifold distribution system from which assimilates are bled off from a central low-resistance pathway via high-resistance leaks, thus making the photosynthates available almost equally throughout the plant. After unloading from the SE/CC complex through plasmodesmata, assimilates follow a symplastic pathway in most sinks, although a later apoplastic pathway may intervene.
In some sink organs part of the phloem unloading pathway is apoplastic. In principle, the apoplastic step could be located at the site of SE/CC complex. An apoplastic pathway is required in developing seeds because there are no symplastic connections between the maternal tissues and the embryo.
Sugars exit the sieve elements (unloading) via a symplastic pathway and are transferred from the symplast to the apoplast at some points removed from the SE/CC complex.
The apoplastic step permits membrane control over the substances that enter the embryo because two membranes must be crossed in the process. In growing maize leaves and sugar beet storage roots the plasmodesmatal frequencies between the SE/CC complex and surrounding cells of the sink are very low.
The parasitic Cuscuta and endomycorrhizae of powdery mildew are intense sinks but do not establish symplastic connections with their hosts. These observations suggest that the rates of trans membrane solute and water transport are exceptionally high for these sieve elements.
It is suggested that either unknown plant membrane transport carriers or porin-like aqueous channels might be involved. As apoplastic solutes lower cell turgor apoplastic unloading has important implications for the control of turgor pressure within a sink.
When phloem unloading is apoplastic, the transport sugar can be partly metabolized in the apoplast, or it can cross the apoplast unchanged. For example, sucrose can be hydrolysed into glucose and fructose in the apoplast by invertase and the derived glucose and fructose would then enter the sink cells. Such sucrose-cleaving enzymes play a role in the control of phloem transport by sink tissues.
Growing leaves, roots, and storage sinks in which carbon is stored in starch and protein molecules utilize symplastic phloem unloading. Transport sugars are used as substrate for respiration and are metabolized into storage polymers and into compounds needed for growth. Sucrose metabolism results in a low sucrose concentration in the sink cells, thus maintaining a concentration gradient for sugar uptake.
No membranes are crossed during sugar uptake into the sink cells, and unloading through the plasmodesmata is passive because transport sugars move from a high concentration in the sieve elements to a low concentration in the sink cells. Metabolic energy is thus required in these sink organs for respiration and for biosynthetic reactions.