Complex Structure and Function of Sieve Elements and Companion Cells

In the March, 2002 issue of Trends in Plant Science (7(3):126-32), Aart JE van Bel, Katrin Ehlers and Michael Knoblauch at the Institute of General Botany, Giessen, Germany, have reviewed the recent progress made in understanding the structure and function of the angiosperm phloem. Sieve elements and companion cells comprise the conducting channels of phloem tissue, and are closely associated to form a single physiological unit. The authors draw attention to the fact that recent non-invasive studies have made it possible to determine what goes on inside intact sieve tubes, allowing study of the processes of mass transport, sieve-pore sealing and conformational changes of structural proteins.

In their introductory remarks, the authors describe how the phloem tissue has developed in angiosperms. In the course of evolution the sieve tube lost its nucleus, the vacuole,Golgi bodies, cytoskeletal elements, ribosomes and a large number of mitochondria. The sieve tube was left with only the plasma membrane, the endoplasm reticulum (ER) and a few enlarged mitochondria. Further conspicuous components are sieve-element plastids which occur in all angiosperms and structural phloem-specific proteins which mainly occur in dicots. Very specialized structural crystalloid protein bodies are found only in legume sieve elements. Both the sieve element and the companion cell are derived from the division of a common mother cell, each with a nucleus and other cytoplasmic organelles. During the course of development, the nucleusof the SE disinte- grates together with the breakdown of other organelles.

The symplasmic connections between SE and CC enable the enucleate sieve elements to survive for long periods of time.

The xylem and the phloem components of the vascular tissue transport water and solutes in opposite directions. The movement of xylem contents flows from the root to the leaf upwards and laterally; in the phloem tissue, on the other hand, the mass flow occurs in the opposite direction, from the leaf to the organs located beneath and laterally. The SEs need a plasma membrane lining to set up the gradients generating a mass flow against the transpiration. This is in contrast to the xylem tissue, where the mass flow is generated by a difference in water potential between soil and air (transpiration flow).

Objections were raised to the concept of mass flow through the sieve plates because of the presence of large protein deposits and the ER near the sieve plates, which were thought to block movement of materials in planta. These deposits were later determined to be artifacts caused by sample preparation for electron microscopy. Recent observations using Confocal Laser Scanning Microscopy (CLSM), aided by the use of fluorochromes, provide evidence that mass flow occurs through sieve pores in spite of the presence of protein deposits and ER on the sieve plates. Other techniques have been developed to measure water flow through sieve tubes in real time. Recently, nuclear magnetic resonance (NMR) has been used for in vivo determination of flow rates and identification of solutes.

Recent studies have shown that macro- molecular extensions anchor the organelles to each other and to the plasma membrane. Because of this anchorage, the cytosolic components are retained within the sieve element, only allowing trafficking of numerous classes of soluble molecules.

An examination of the sieve-element sap has revealed the presence of about 200 phloem-specific proteins, which are referred to as sieve tube exudate proteins (STEPs). The functions of some these proteins, the majority of which may be produced in companion cells, have been determined. This suggests that STEPs are transported from companion cells to sieve elements.

P-proteins are structural proteins that develop in sieve elements in most species with the exception of most palms and grasses. P-protein bodies disperse at maturity and are located near the wall in mature sieve elements. The sieve elements of Vicia faba have 10-30 micrometer-long giant crystalloid protein bodies, which are P-proteins that appear to belong to a different class than those found in other families. These structures, if destroyed by micro-capillary tip, reform in a matter of a few seconds.

Calcium ions can induce a conformation change of these crystalloid protein bodies. Turgor pressure changes are thought to trigger short signaling chains that induce release of calcium ions from the apoplast or from the ER, suggesting a mechanism for the dispersal and re-formation of crystalline phloem protein structures in which calcium is engaged. One of the functions of dispersed crystalline phloem proteins in legumes may be to plug the sieve pores in the event of injury.

The sieve elements are characterized by the universal presence of plastids. These plastids may have starch inclusions, protein inclusions, both starch and proteins, or neither type. Sieve element plastid ultrastructure is characteristic of each plant family, and thus has been used for systematic and evolutionary studies. The plastids seem to have greater structural
integrity than P-protein bodies, as plastids are capable of withstanding greater mechanical force before rupturing. The function of sieve element plastids is not known.

Callose has been proposed to serve as the major sieve-pore sealant in vivo. However, phloem-specific proteins have more recently been suggested to perform sieve pore plugging immediately after wounding. Apart from the visual evidence in favor of instant sieve pore plugging by phloem- specific proteins, there is some circumstan- tial evidence against callose being an important sealant in immediate sieve pore plugging.

Callose is often formed in response to wounding, and thus callose detected in electron microscopy sections are often suspected to result from cutting and fixation. Callose is involved in the origin of sieve pores and is also seen around the mature sieve pores. Callose is thought to be involved in closure of plasmodemata, structures that are homologous to the sieve pores, but it remains to be determined whether callose synthesis is rapid enough for the instantaneous occlusion that occurs following wounding.

The authors describe the unique structure of plasmodesmata connecting companion cells and sieve elements. These structures have one larger channel at the sieve element side and a number of narrower plasmodesmal branches on the side of the companion cell. This structure is called a pore-plasmodesma unit (PPU) to differentiate it from plasmodesmata found in other tissues. It was shown in the 1990s using radiolabeled methionine that sieve-element protein turnover takes place in the companion cell. The commonly accepted molecular exclusion limit of plasmodesmata at that time was thought not to exceed 1 kDa. However, intracellular injection of
fluorescent macromolecules showed that the PPU exclusion limit is between 10 kDa and 40 kDa. It was also shown that the monomeric 24 kDa lectin PP2 can move between the sieve elements and companion cells of Cucurbita maxima. By using the 27 kDa green fluorescent protein, it was demonstrated that the transport of proteins occurs from the site of synthesis in companion cells to the sieve elements and from there to sink tissues. Recent studies in macromolecular trafficking have shown that certain STEPs, such as thioredoxin h from rice and classes of phloem-specific proteins from Ricinus communis and Cucurbita maxima, are capable of increasing the molecular size exclusion limit (SEL) of the plasmodesmata between mesophyll cells. This was demonstrated using tobacco mesophyll cells in which the SEL was increased to 9-20 kDa by rice thioredoxin h. The function of factors such as these may be to keep the PPU in a widened state.

The mechanism by which STEPs are transported from companion cell to sieve element is still unknown, but it has been noted that many STEPs are small and acidic, although some STEPs have a molecular size exceeding 30 kDa. Heat-shock proteins have been suggested to be involved in trafficking STEPs through dilated PPUs. Recently proposed models have included docking proteins, chaperone proteins and translocating proteins, possibly interacting with the ER tubule that runs through the PPU.

Some phloem-specific proteins may exert remote control on distant tissues. The 18-amino acid systemin, probably synthesized in the companion cells, is released into the sieve elements and induces the production of proteinase inhibitors as a weapon against insect predator attacks on leaves. Structural P-proteins may also migrate long distances. By means of graft experiments involving two genera of the Cucurbitaceae, it has been shown that stock specific proteins travel from the stock to the scion as shown by the analysis of sieve-tube exudates.

In addition to long-distance transport of proteins through the phloem, mRNA transcripts have also been found to traffic through PPUs. Transfer of mRNAs encoding the sucrose carrier SUT1, thio- redoxin h, oryzacystatin and actin, and CmNACP has been reported in studies on potato, rice and cucurbits. Ribosomes are absent in mature sieve elements, and thus it is not thought that mRNAs in the sieve-element sap are translated locally. Rather, transport of mRNAs is thought to indicate remotely control gene expression in other parts of the plant. Questions remain regarding whether the release of proteins and transcripts into the sieve elements is a regulated process, as well as what the exact mechanisms of transport are.

Many plant viruses are capable of moving through plasmodesmata, often after dilating the plasmodemata with movement proteins. The authors discuss how studies on the passage of viruses such as geminivirsuses and luteoviruses through PPUs may shed light on the mechanism of RNA trafficking through PPUs. Geminiviruses and luteoviruses are unable to pass through the plasmodesmata between companion cells and phloem parenchyma and are therefore contained within the sieve tubes. Current studies are focusing on these viruses as a tool for understanding the mechanism of macromolecular trafficking through the PPUs.

In light of the recent achievements in unraveling some of the mysteries surrounding the sieve element-companion cell complex, the authors express confidence that more secrets about this structure will be elucidated through the application of new techniques.

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