Susan Dunford, University of Cincinnati, Cincinnati, OH, USA
Revised by Alexander Schulz, University of Copenhagen,
Methods for demonstrating that sugar is transported in sieve elements
In classic experiments on the translocation of organic solutes performed by the Italian anatomist Marcello Malpighi in 1686, the bark of a tree was removed in a ring around the trunk (Web Figure 12.1.A). In 1928, T. G. Mason and E. J. Maskell observed that this treatment, called girdling, has no immediate effect on transpiration, since water moves in the xylem, interior to the bark (Mason and Maskell, 1928). However, sugar transport in the trunk is blocked at the site where the bark has been removed. Sugars accumulate above the girdle—that is, on the side toward the leaves—and are depleted below the treated region.
Eventually the bark below the girdle dies, while the bark above swells and remains healthy. Mason and Maskell concluded that sugar is transported in the bark of the tree and that the sieve elements are the cellular channels of sugar transport. The latter conclusion was based on an observed high correlation between leaf and bark sucrose contents and on the high sucrose concentration calculated to be present in the sieve elements.
More sophisticated experiments on phloem translocation became possible in the 1940s, when radioactive isotopes became available for scientific research. Labeled organic compounds can be introduced into the plant in various ways. For example, carbon dioxide can be labeled with 14C or 11C and supplied to an intact mature leaf enclosed in a sealed chamber. These two isotopes of carbon, 11C and 14C, have very different half-lives (the half-life is the time required for the radioactivity to decrease by one-half). The half-life for 11C is 20 min; that for 14C is 5,600 years. For this reason, the isotopes are used in different types of experiments. Carbon-14 is useful for long term experiments analyzing the compounds in which the radioactivity is present. Carbon-11 would disappear before such an analysis could be made. Carbon-11 is most useful when the researcher wishes to repeat measurements on the same plant. Since only a few hours are needed for the isotope in the plant to disappear, the radioactivity used in one experiment does not interfere with next.
The labeled carbon dioxide is incorporated into organic compounds via the CO2-fixing reactions of photosynthesis (see Web Topic 10.2) and eventually into transport sugars. In another type of experiment, researchers bypass the photosynthetic reactions by supplying labeled transport sugar, usually sucrose, directly to the leaf.
To identify the pathway of sugar translocation, we must determine the tissue or cellular location of the isotope, usually by means of a technique called autoradiography. In tissue autoradiography, tissue of labeled plants is rapidly frozen, freeze-dried, embedded in paraffin or resin, and sliced into thin sections. The sections are then coated with a film of photographic emulsion.
During a period of exposure, radiation from the radioactive compound fed to the plant exposes the film, and when the film is developed, silver grains appear wherever the label was present in the tissue. A comparison of the tissue section and the pattern of silver grains shows the location of the label in the tissue. In terms of the transport pathway for sugars, the label initially appears in the sieve elements of the phloem, confirming the results of the earlier experiments (Web Figure 12.1.B).
Anatomical and developmental factors affecting source to sink patterns
Phloem transport occurs in sieve elements from sources to sinks. Although the overall pattern of transport in the phloem can be stated simply in this way, the specific pathways involved are often more complex, depending on proximity, development, vascular connections, and modification of translocation pathways. Not all sources supply all sinks on a plant; rather, certain sources preferentially supply specific sinks. In the case of herbaceous plants, such as sugar beet (Beta vulgaris) and soybean (Glycine max), the following generalizations can be made:
- The proximity of the source to the sink is a significant factor. The upper mature leaves on a plant usually provide photosynthates to the growing shoot tip and young, immature leaves; the lower leaves supply predominantly the root system. Intermediate leaves export in both directions, bypassing the intervening mature leaves.
- The importance of various sinks may shift during plant development. Whereas the shoot and root apices are usually the major sinks during vegetative growth (and in that order), seeds and fruits generally become the dominant sinks during reproductive development, particularly for adjacent and other nearby leaves.
- Source leaves preferentially supply sinks with which they have direct vascular connections (Web Figure 12.1.C), whether those sinks are young leaves or roots. In the shoot system, for example, a given leaf is generally connected via the vascular system to other leaves directly above or below it on the stem. Such a vertical row of leaves is called an orthostichy. The number of internodes between leaves on the same orthostichy varies with the species.
- Interference with a translocation pathway by wounding or pruning can alter the patterns established by proximity and vascular connections that have been outlined here. In the absence of direct connections between source and sink, vascular interconnections, called anastomoses (singular anastomosis) can provide an alternative pathway. In sugar beet, for example, removing source leaves from one side of the plant can bring about cross-transfer of photosynthates to young leaves (sink leaves) on the pruned side (see Web Figure 12.1.C). Removal of the lower source leaves on a plant can force the upper source leaves to translocate materials to the roots, and removal of the upper source leaves can force lower source leaves to translocate materials to the upper parts of the plant.
The plasticity of the translocation pathway depends on the extent of the interconnections between vascular bundles and thus on the species and organs studied. In some species, the leaves on a branch with no fruits cannot transport photosynthate to the fruits on an adjacent defoliated branch. But in other plants, such as soybean, photosynthate is transferred readily from a partly defruited side to a partly defoliated side.
As a proxy for the use of radioactive assimilates, carboxy fluorescein diacetate and its photoactivatable derivatives have been used to visualize the phloem network and the pre-phloem pathway. Thus, continuity of symplasmic pathways have been proven, both via plasmodesmata, sieve pores, and/or sieve-pore plasmodesma contacts (Liesche and Schulz, 2012). These techniques work, however, only in the intact phloem system and depend on the use of confocal microscopy that makes thin optical sections in an otherwise intact tissue (see Web Figure 12.1.D).
Joy, KW (1964) Translocation in Sugar-Beet: I. Assimilation of 14CO2 and Distribution of Materials from Leaves. Journal of Experimental Botany 15: 485–494.
Liesche J, Schulz A (2012) In Vivo Quantification of Cell Coupling in Plants with Different Phloem-Loading Strategies. Plant Physiology 159: 355–365.
Mason TG, Maskell EJ (1928) Studies on the Transport of Carbohydrates in the Cotton Plant: II. The Factors determining the Rate and the Direction of Movement of Sugars. Annals of Botany Vol os-42: 571–636.