Use of Phloem Exudate Technique in the Study of Amino Acid Transport in Pea Plants

Abstract

The phloem exudation technique using ethylenediaminetetraacetic acid (EDTA) was evaluated In studies of amino acid translocation in Pisum sativnm L. seedlngs. Exudation of phloem sap from cut petioles of fuily expanded leaves was enhanced by EDTA (20 millimolar disodium salt [pH 7.01). Amino acids (mainly asparagine, homoserine, glutamate, and also aspartate and serine) were present in petiole exudates from EDTA-treated leaves at levels which were commonly 5-to 10-fold (or more) higher compared with water-treated controls. Exudation was greater from darkened leaves, and the pattern of amino acids was markedly different from the more uniform mixture leaking from water-treated controls.After feeding 'C-labeled amino acids to the leaf blade, distribution of radioactivity in components of the exudate differed from that of the leaf tissue, suggesting selectivity of amino acid loading. 1'ClAsparagine was converted to 2-hydroxysuccinamic acid and to other amino acids by the leaf, but was recovered in exudate mainly as asparagine (60%) and aspartate (30%). SImilarly, in the exudate, 65 to 70% of the label from 114CI-aspartate was in amino acids, although in the leaf tissue 50% was in the organic acid fraction and only 11% remained as aspartate. Metabolism of asparagine and aspartate was essentially the same in intact leaf blades as in EDTA-treated leaves. Despite the possibility of EDTA damage in the petiole, phloem loading of amino acids appeared to be relatively unimpaired.Although the amount of labeled material appearing in the exudate is less than the amount translocated in the intact plant, the technique is useful in the study of amino acid transport.Mature, transpiring leaves import nitrogen from the roots, with varying proportions of nitrate and organic nitrogen, depending on species and growth conditions (17). In Pisum sativum, the nitrogen level of the growing leaves stabilizes when the leaves are fully expanded (3), thus most of the incoming nitrogen must be reexported. In a continuing study of amino acid metabolism and transport in peas (3), the redistribution of amino acids in phloem has been investigated.Several methods have been used in the past to obtain phloem samples from leaves. Phloem sap was collected from severed inflorescences of palms and some Agavacae (22) Phloem sap has been collected from the severed stylets of aphids (10) and amino acids were detected in aphid stylets exudate from willows (16,20) and from peas (2). The quantities of sap obtained are small and the difficulty in prepositioning the aphids limit the usefulness of this method in such studies.None of these techniques seemed suitable for collection of the phloem sap exported from fully expanded leaves of vegetative plants. Although some leaves do exude a phloem sap through the cut petiole (12), the quantities thus obtained are minute. King and Zeevaart (1 1) described an EDTA-promoted exudation of phloem sap from detached leaves. The method has subsequently been used with a variety of plants by other research groups (6,8,9,...

Reversible Calcium-Regulated Stopcocks in Legume Sieve Tubes [W]

“…The effect is commonly ascribed to the inhibition of Ca 2ϩ -dependent stimulation of callose synthesis (Sabnis and Sabnis, 1995). However, the moderate Ca 2ϩ -mediated increase of callose formation on SPs detectable after hours of treatment (King and Zeevaart, 1974) does not seem to provide a sufficient explanation for the dramatic stimulation of phloem exudation by chelating agents (Fellows et al, 1978; Urquhart and Joy, 1981;Schulz, 1998). Our results demonstrate that an additional Ca 2ϩ -dependent mechanism operates in the Fabaceae.…”

Comprehensive Dissection of Spatiotemporal Metabolic Shifts in Primary, Secondary, and Lipid Metabolism during Developmental Senescence in Arabidopsis

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Molecular and Functional Analyses Support a Role of Ornithine-δ-Aminotransferase in the Provision of Glutamate for Glutamine Biosynthesis during Pine Germination

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