5.4.3  Chemical nature of translocated material

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(a)  Techniques to collect phloem sap

Since phloem translocation is confined to sieve elements embedded within a tissue matrix, it is difficult to obtain uncontaminated samples of translocated sap. The least equivocal approach has been to take advantage of the high P of sieve tube contents. Puncturing or severing sieve tubes should cause exudation of phloem sap provided a sealing mechanism is not activated.

For some plant species, sieve-pore sealing (Section 5.4.2(c)) develops slowly, or can be experimentally down-regulated by massage or repeated excisions (Milburn and Kallarackal 1989). Carefully placed incisions that do not disturb the underlying xylem of these species permit collection of relatively pure phloem sap exuded through severed sieve tubes (see Case study 5.1). The excision technique has been expanded to plant species that do not readily exude, by chemically inhibiting the sealing mechanism. Callose production is blocked when wounded surfaces are exposed to the chelating agent ethylenediaminetetraacetic acid (EDTA) by complexing with calcium, a cofactor for callose synthetase. Immersing whole, excised organs in EDTA solution, which is essential to inhibit blockage, risks contaminating sap with solutes lost from non-conducting cells. This is not an ideal technique.

Enlisting sap-sucking aphids to sample sap has been more successful. Aphids can guide a long syringe-like mouthpart (a stylet) into conducting sieve elements (Figure 5.19). Pressure normally forces sieve-tube sap through the stylet into the aphid’s gut where it becomes food or is excreted as ‘honeydew’. By detatching the aphid from its mouthpart pure phloem sap can be collected from the cut end of the implanted stylet.


Figure 5.19 Aphids can be used to collect phloem sap. Top photograph: a feeding aphid with its stylet embedded in a sieve tube (see insert); scl, sclerenchyma; st, stylet; x, xylem; p, phloem. Note the drop of ‘honeydew’ being excreted from the aphid’s body. Plates (a) to (e) show a sequence of stylet cutting with an RF microcautery unit at about 3-5 s intervals (a to d) followed by a two-minute interval (d to e) which allowed exudate to accumulate. The stylet has just been cut in (b); droplets of hemolymph (aphid origin) are visible in (b) and (c); once the aphid moves to one side the first exudate appears (d), and within minutes a droplet (e) is available for microanalysis. Scale bars: top = 1 mm, bottom = 1.5 mm (Courtesy D. Fischer)

(b)  Chemical analysis of phloem sap

Chemical analyses of phloem sap collected from a wide range of plant species have led to a number of generalisations (e.g. Milburn and Baker 1989). Phloem sap is a concentrated solution (10–12% dry matter), generating an osmotic pressure (Π) of 1.2 to 1.8MPa. Sap pH is characteristically alkaline (pH 8.0 to 8.5). The principal organic solutes are non-reducing sugars, amides (glutamine and asparagine), amino acids (glutamase and aspartase) and organic acids (malate). Of these solutes, non-reducing sugars generally occur in the highest concentrations (300–900mM). Nitrogen is transported through the phloem as amides and amino acids; nitrate is absent and ammonium only occurs in trace amounts. Calcium, sulphur and iron are scarce in phloem sap while other inorganic nutrients are present, particularly potassium which is commonly in the range of 60–120mM. Physiological concentrations of auxins, gibberellins, cytokinins and abscisic acid have been detected in phloem sap along with nucleotide phosphates. The principal macromolecule is protein, comprised largely of P-protein in dicotyledons, but a number of enzymes are also detectable.

(c)  Significance of the chemical forms translocated

Phloem sap provides most inorganic and all organic substrates necessary to support plant growth (see Case study 5.1). Non-transpiring tissues are particularly dependent on resources delivered in the phloem (Section 5.3.1). That translocated sugars represent the major chemical fraction of the phloem sap (see Section 5.4.3(b)) is consistent with the bulk of plant dry matter (90%) being composed of carbon, hydrogen and oxygen. Carbon transport is further augmented by transport of nitrogen in organic forms.

Carbohydrate is translocated as non-reducing sugars in which the metabolically reactive aldehyde or ketone group is reduced to an alcohol (mannitol, sorbitol) or combined with a similar group from another sugar to form an oligosaccharide. Apart from sucrose, transported oligosaccharides belong to the raffinose series. In this series, sucrose is bound with increasing numbers of galactose residues to form raffinose, stachyose and verbascose respectively. However, sucrose is the most common sugar species transported. In a small number of plant families, other sugar species predominate. For example, the sugar alcohol sorbitol is the principal transport sugar in the Rosaceae (e.g. apple) and stachyose predominates in the Cucurbitaceae (e.g. pumpkin and squash). Exclusive transport of non-reducing sugars probably reflects packaging of carbohydrate in a chemical form which protects it from being metabolised. Metabolism of these transported sugars requires their conversion to an aldehyde or ketone by enzymes which are absent from sieve-tube sap.