Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Solute Transport:  Phloem Structure & Function

I. Definition
    Solute transport in plants, translocation, primarily occurs in the phloem, but it can occur in the xylem. 

II. Solute Transport in the Xylem

• Some solutes are transported in the xylem
• Water and dissolved ions are the main substances in vessels/tracheids
• These materials are transported via transpiration stream
• Xylem sap may also contain organic materials, usually in relatively low concentration (with a notable exception being maple sap in the spring which is comprised of 2% or more sucrose).  See table on overhead.
• Substances move at different rates depending on matrix effects, metabolic needs, etc.

III. Solute Transport in the Phloem

1. Phloem is difficult to study in plants because: (1) the transport cells/tissue in plants are small (microscopic) in comparison to the transport structures in animals; (2) there is a very rapid response of the phloem to wounding (contents under pressure); (3) transport in plants is intracellular (vs. extracellular in animals); and (4) the transport cells are alive.
   
2. Phloem is the primary transport tissue for photosynthates (photoassimilates, or simply stated - organic materials). 
       Radiotracer studies in which leaves are briefly exposed to ¹⁴C-labeled carbon dioxide show that radioactive photosynthates are localized in the phloem.
   
3. Aphids Don't Suck
       Kennedy & Mittler (1953) first noted that aphids could be used as a direct pipeline to the phloem.  Phloem-feeding aphids stick their hollow, syringe-like stylet directly into phloem cells.  Surprisingly, the phloem doesn’t seal itself in response.  Aphids don't suck; rather, the phloem contents are forced into the aphid (thus the phloem is under pressure) and the excess oozes out the anus (honeydew). Thus, aphid studies demonstrate that the phloem is under pressure.  Further, the honeydew can be collected and we can identify its composition. Better yet, after anaesthetizing the aphid with CO₂ the body is severed from the stylet leaving a miniature spile tapped directly into the phloem.
   
4. Phloem Content (see table on overhead)
       Analysis - early studies to determine the content of the phloem involved cutting into the plant and analyzing the contents of the sap that was recovered.  The problem is that you couldn't be sure that your sample wasn't contaminated by xylem exudates or other materials.  Aphid studies described above helped to solve this problem.  Phloem is rich in:
   
   1.  Carbohydrates - make up 16-25% of sap. The major organic transport materials are sucrose, stachyose (sucrose-gal), raffinose (stachyose-gal). These are excellent choices for transport materials for two reasons: (a) they are non-reducing sugars (the hydroxyl group on the anomeric carbon, the number one carbon, is tied up) which means that they are less reactive and more chemically stable; and (b) the linkage between sucrose and fructose is a "high-energy" linkage similar to that of ATP. Thus, sucrose is a good transport form that provides a high energy, yet stable packet of energy; 
   
   2. Amines/amides (0.04-4%) such as asparagine, glutamine, aspartic acid, ureides like ureas, citrulline, allantoin and allantoic acid. These compounds serve to transport "nitrogen"; 
   
   3. ATP, hormones, sugar alcohols like sorbitol (apple, pear, prune) and mannitol (mangrove, olive), and an assortment of other organic materials; and 
   
   4. Inorganic substances including magnesium and potassium.
   
5. Direction of phloem transport 
       Information derived from several experiments; check out the Phloem Case Studies.
   
   (1) Classic girdling experiments (removing the bark of a woody plant) by Malphigi (1675) and Hales (1725) provided some of the earliest evidence. These experiments showed the accumulation of material above the girdle, and that carbohydrates were not translocated below the girdle. Thus, plants transport substances in the phloem downward toward the roots.
   
   (2) Sophisticated girdling experiments, using tracers like ³²P, ¹³C, and ¹⁴C demonstrate that substances in the phloem are transported downward towards the roots OR upwards toward the shoot meristem. See data on overheads.
   
   (3) Aphids and tracers (see overhead)
   
   Conclusions - phloem transports organic materials from sites of production (called a source) to a site of need (called a sink). Thus, the typical direction of transport is downward from the primary source (leaves) to the major sink (roots). 
    
6. Rate of phloem transport
       Aphid experiments once again provide an answer...translocation rates average about 30 cm hour^-1 or even faster.
    
7. The phloem is under pressure
       Studies with aphids showed that the sap was "pushed" out of the plant suggesting the phloem is under pressure.  More recent studies with sophisticated pressure probes have shown a pressure gradient from source to sink.

IV. Phloem Anatomy

A. Cell types.

1. Sieve tube members or sieve elements.
       These cells are joined end to end to make a sieve tube. Theye are called sieve cells in gymnosperms. At maturity, these cells: (a) are alive, (b) have a functional plasma membrane and therefore are osmotically active/responsive; (c) no tonoplast or vacuole; (d) no nucleus, thus no DNA-directed protein synthesis, (e) few mitochondria or plastids; (f) the ER is primarily beneath plasma membrane and it is mostly smooth.

    Sieve elements are joined by sieve plates. These have numerous pores lined with callose (β 1-3 glucan). Callose forms rings around the pore, like a grommet. The wall region in the middle of the grommet hollows out and the membranes from the two adjacent cells are connected. Callose can plug the pore if the cell is damaged. The amount of callose observed varies with season, age, metabolism. Callose synthase is in the cell membrane.


2. Companion cells (angiosperms; albuminous cells - gymnosperms)
       These cells have a dense cytoplasm, mitochondria, nucleus, golgi, ER, chloroplasts - the standard goodies. Although their function is not well understood, they can be considered "nurse cells" to the sieve tube members. These cells are derived from the same cambial initial cell as the sieve tube members. There are three types of companion cells:
   
   (a) "ordinary" – with chloroplasts, few plasmodesmata connections to other cells except sieve elements, smooth inner walls, normal chloroplasts;
   
   (b) transfer – more plasmodesmata, ingrowths in the wall to increase the S/V ratio; and
   
   (c) intermediary – many plasmodesmata, vacuoles, undeveloped chloroplasts.  The transfer and "ordinary" companion cells likely function to remove solutes from the apoplast
   
3. Parenchyma cells
       These are vacuolated, storage cells. They help in lateral conduction and may help in transferring material to/from sieve cells. Transfer cells are specialized parenchyma cells.
    
4. Fibers - primarily for support.
    
5. Side note:  Mesophyll cells in a leaf are close (perhaps 1-3 cells away) to a minor vein.

• MW 14,000-158,000
• Originally thought to be a carbohydrate and was called slime because it gelled when exposed to the air
• Various forms, bundles of fibers or amorphous areas or even crystalline
• Appear early in development of sieve elements
• Only in angiosperms
• at least two proteins, PP1 and PP2
• Once the sieve pores form, the P-protein disperses through the pore.
• The protein is fibrous
• P protein plugs the pore when the cell is damaged.
• synthesized in companion cells

VI. Mechanism for phloem transport

1. Requirements
       The model must account for: (1) speed of transport. The process is much faster than simple diffusion. For example, a conservative estimate of the mass transfer rate in phloem is 15 g cm^-2 hr^-1. If the rate was based solely on diffusion is would be predicted to be 200 μg cm^-2 hr^-1; (2) bidirectional flow - recall that substances can be transported down or up in the phloem; and (3) pressures in the phloem
    
2. Pressure flow (or Bulk Flow) hypothesis of Munch. This is the best model that fits the data.  
   
       The Model: Phloem transport is analogous to the operation of a double osmometer (see diagram). If solute is added to bulb A → osmotic potential decreases → osmotic uptake of water → pressure increases → bulk flow of water and solute to bulb B → pressures increases in bulb → water potential in B greater than in beaker → osmotic flow of water into the beaker → water returns to side A via the connection. This system could be maintained indefinitely if there is a mechanism to remove solute (sucrose) at the end (sink) and a mechanism to add solute (source).

    Sinks include young leaves, roots, developing fruits.  Sources include mature leaves, cotyledons, endosperm, and bulbs and storage roots in spring.  Sinks and sources can change depending upon the nutritional need of the plant.  Thus, roots can be a source in the spring but are sinks for the majority of the growing season.
 

3. Plants as osmometers. If this model is valid then.....

1. Sieve tubes should be continuous pipes...they are.
   
2. Sieve tubes should provide minimal resistance to flow. 
       In other words, the sieve tubes shouldn�t be clogged by P-protein. This is true in specimens that are rapidly prepared. However, this was a major concern in early experiments because the phloem always appeared clogged up in TEM pictures.  Further, this explains why sieve tube members have few "typical" cellular structures - they would "get in the way."
   
3. The phloem should be under pressure.  As the aphid experiments suggest....it is. 
       In fact, mini-pressure gauges can be attached to a severed aphid stylet and the pressure can be measured.  It varies from 0.1-2.5 MPa. Further, there should be a pressure gradient from source to sink (driving force for movement). There is...see overhead.
   
4. Sieve elements must have a membrane (for development of pressure gradients) - they do.
   
5. There should be an osmotic potential gradient from source to sink (there is...see overhead). 
       The source region of the phloem has a considerably lower osmotic potential than the sink regions.
   
6. There must be a mechanism to load solutes from the source into sieve cells. 
       This process must be active since the solutes (usually sucrose) are being loaded against a concentration gradient. Evidence - respiratory inhibitors block the process. The loading mechanism should be:

• Selective - it should only load the materials that are transported. This is supported by radiotracer studies; abraded leaves have been shown to only load materials that are normally transported;

• Allow for apoplastic (from protoplast to wall to protoplast) or symplastic (from protoplasts to protoplast via plasmodesmata) transport.  In some species, sucrose transport is symplastic - from mesophyll protoplast to cc-se protoplast via plasmodesmata. In others, sucrose loading into the cc-se complex involves an apoplastic step (mesophyll protoplasts to apoplast to cc-se protoplast.

• Provide a mechanism to transport sucrose across the membrane - the sucrose/proton cotransport system. According to this model, protons are pumped out of the sieve cells into the apoplast by a membrane-bound H⁺-ATPase → the proton concentration increases in the apoplast → pH decreases → K⁺ is brought into the sieve cell to balance the charge → the proton gradient provides the driving force for transporting sucrose against a gradient → the sucrose and protons bind to a carrier protein in the membrane and are released in the sieve tube member. Evidence: the pH is high in sieve tubes; if the pH of the apoplast is increased there will be no sucrose uptake; there is a hi potassium conc. in sieve tube members. A membrane carrier is likely involved since PCMBS (p-chloromercuribenzene sulfonic acid), an inhibitor of membrane proteins, interferes with sucrose uptake.

7. There must be a mechanism to unload solute at the sink. Sucrose is unloaded into the apoplast in some tissues (i.e., ovules) and into the symplast of others (growing/respiring tissues like young leaves, meristems).
   
8. Apoplastic transport and unloading can occur via two methods: (a) sucrose is hydrolyzed by acid invertase to glucose and fructose upon reaching the sink. This maintains the gradient for transport. The glucose and fructose are taken up by the sink cells and stored or further metabolized as in maize; or (b) sucrose is unloaded into the sink by a carrier co-transport system like in sucrose loading.
   
9. The empty ovule technique has been useful in these studies.
   
10. Some metabolism is required (for loading/unloading) and to maintain sucrose against a concentration gradient. This explains the response to respiratory inhibitors. Phloem transport is also inhibited by anoxia and cold temperatures - both thought to exert their effect through energy metabolism.

VII. Problems with the model
    Bidirectionality - how can phloem translocate materials in two different directions at once? It can’t, at least not within the same sieve tube. However, presumably sieve tubes within a single vascular bundle could be transporting in opposite directions assuming each is acting appropriately.

Last updated:  01/07/2009     � Copyright  by SG Saupe