Postharvest Biology and Technology 18 (2000) 81 – 84
Short communication
Reversal of glyphosate inhibition of
Sandersonia aurantiaca
flower senescence with aromatic amino acids
Jocelyn R. Eason
a,*, Jason W. Johnston
b, Leigh de Vre´
aaNew Zealand Institute for Crop & Food Research Limited,Food Industry Science Centre,Batchelar Road,Pri6ate Bag11 600,
Palmerston North,New Zealand
bMassey Uni6ersity,Pri6ate Bag11 222,Palmerston North,New Zealand
Received 21 June 1999; accepted 26 August 1999
Abstract
Glyphosate (N-(phosphonomethyl) glycine) is a broad spectrum post-emergence herbicide. This herbicide inhibits the shikimate pathway enzyme EPSP synthase (5-enol pyruvylshikimate 3-phosphate synthase), thereby interfering with aromatic amino acid metabolism. During preliminary investigations with inhibitors of protein and amino acid biosynthesis, we noticed that vase solutions containing glyphosate altered the normal pattern of Sandersonia aurantiacaflower senescence. Further studies showed that although glyphosate (2 mM) was toxic to all green tissue on the flower stem, the senescence of mature flowers (no green tissue) was delayed. Glyphosate-treated flowers did not fade but stayed a bright orange colour and the compressive strength of the flowers was greater (the flowers were less wilted) than the control flowers that were held in water. Treatment of flowers with vase solutions of phenylalanine (2 mM) and tyrosine (2 mM) in the presence of glyphosate reversed the beneficial effect that glyphosate treatment had on flower senescence. The data indicate that a lack of aromatic amino acids may be the cause of delayed fading and wilting of glyphosate-treated sandersonia flowers. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Flower; Senescence;Sandersonia aurantiaca; Glyphosate; Phenylalanine; Tyrosine
www.elsevier.com/locate/postharvbio
1. Introduction
The visible signs of flower senescence — colour changes, wilting, abscission — are relatively late events in the process of senescence. We have
previously demonstrated that the visible symp-toms of senescence in Sandersonia aurantiaca
flowers are preceded by the exhaustion of storage carbohydrate (starch) and a reduction in the levels of soluble carbohydrates and soluble proteins (Ea-son et al., 1997).
We have been studying the effect of inhibitors of protein and amino acid biosynthesis on flower senescence (Eason and de Vre´, 1995). In this * Corresponding author. Tel.: +64-6-356-8300; fax: +
64-6-351-7050.
E-mail address:[email protected] (J.R. Eason)
J.R.Eason et al./Posthar6est Biology and Technology18 (2000) 81 – 84 82
paper we report on the results of trials using the herbicide glyphosate. The major target of this herbicide is EPSP synthase (5-enol pyruvylshiki-mate 3-phosphate synthase, EC 2.5.21.19), an en-zyme in the shikimate pathway. The aromatic amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan are end-products of the shikimate pathway. Previous researchers found that aro-matic amino acids reversed glyphosate toxicity indicating that glyphosate was interfering with aromatic amino acid metabolism (Gresshoff, 1979; Killmer et al., 1981). Although this phe-nomenon has been found in a variety of microor-ganisms and cultured plant cells, it has met with limited success in intact plants (Amrhein, 1986). Glyphosate has been reported to inhibit other enzymes of the shikimate pathway (e.g. 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and dehydroquinate synthase) as a result of the metal-chelating properties of the herbicide (Am-rhein, 1986). Our treatment of flowers with two of the most soluble aromatic amino acids (Phe, Tyr) in the presence of glyphosate was undertaken to determine whether the glyphosate-induced delay of flower senescence was due to a reduction in the pool of aromatic amino acids in the senescing tissues.
2. Materials and methods
2.1. Plant material
Sandersonia tubers (S. aurantiaca Hook.) were obtained from Ocean Flowers (Whangarei, New Zealand) and grown as previously described (Ea-son and Webster, 1995). Flowering stems or indi-vidual mature flowers (stage 7, Eason and Webster (1995)) were cut from plants in the glasshouse and transported with their cut ends in water to the laboratory. The flowers were placed in vase solutions and kept in a controlled environ-ment (12 h light cycle at 2092°C, relative humid-ity 50 – 60%, a light level at bench height of 20 – 25
mE m−2 s−1 provided by cool white fluorescent
tubes).
2.2. Inhibitor treatments
Flowering stems were treated with 2 and 20 mM glyphosate (Roundup®, Monsanto) or held in water. The leaves, stem and flowers were scored daily for signs of phytotoxicity (e.g. wilting, necrosis).
In two separate trials detached mature flowers (stage 7) were held continuously in one of five solutions as follows: (1) deionized water; (2) Phe (1 mM, Sigma), Tyr (1 mM, Sigma); (3) glyphosate (2 mM); (4) glyphosate (2 mM), Phe (1 mM), Tyr (1 mM); and (5) glyphosate (2 mM), Phe (2 mM), Tyr (2 mM). The flowers were scored daily for colour (hue angle) and firmness. The treatments were replicated on four or five flowers. The experiments were carried out two separate occasions with similar results.
2.3. Flower colour and firmness
Flower colour was measured with a Chroma meter (Minolta CR-200) on opposite sides of each flower at the point where the flower diameter is the greatest. Results are expressed as hue angle (H0
=tan−1
b/a, Little (1975)). Flower firmness was measured by compression using an Instron Universal Testing Machine (model 4301). A flat probe (25×5 mm) compressed the flower at its widest diameter by 20% of the original floral diameter. The crosshead speed was 50 mm min−1. Resistance to compression was measured as the maximum load (Newtons).
2.4. Statistical analysis
J.R.Eason et al./Posthar6est Biology and Technology18 (2000) 81 – 84 83
3. Results and discussion
Preliminary investigations revealed that vase so-lutions of glyphosate (2 and 20 mM) were toxic to green tissue, resulting in the early wilting and necrosis of leaves and flower buds on the flower
stems. Treating detached stage 5 flowers with glyphosate also resulted in early wilting and ne-crosis, especially at the tepal tips which were green when the treatment began. Subsequent feed-ing trials were carried out on detached mature pre-senescent flowers (stage 7, no green tissue).
The first visible sign of sandersonia flower senescence is tepal fading from bright orange to pale yellow (indicated by an increase in the hue angle from approximately 68 to 75, Fig. 1). The colour change of flowers held in a solution of Phe and Tyr (1 mM each) was similar to the control flowers (Fig. 1). Glyphosate treatment of flowers inhibited tepal fading, the flowers being a bright orange colour on day 5 (hue angle 570) while the control flowers were pale yellow (hue angle approximately 75). The fading of flowers held in a solution of glyphosate with 1 mM Phe plus 1 mM Tyr was similar to that of the glyphosate-treated flowers (Fig. 1). In a subsequent trial detached stage 7 flowers were held in water, glyphosate or glyphosate with 2 mM Phe plus 2 mM Tyr (Fig. 2). The inhibition of senescence by glyphosate was consistent with the first trial. However, the greater concentration of aromatic amino acids (2 mM) in the vase solution resulted in a pattern of senes-cence for these flowers that was closer to that of the control flowers which were held in water (Fig. 2).
The second visible sign of sandersonia flower senescence is wilting. Tepal wilting was measured by a decline in the compressive strength of the flowers (Fig. 3). The compressive strength of glyphosate-treated flowers was greater than the water controls after day 2 (Fig. 3). Indeed, the decline in compressive strength of these flowers was less over the 5 day treatment period than the controls. The addition of 1 mM Phe plus 1 mM Tyr to glyphosate delayed wilting in a similar manner to flowers treated with glyphosate alone (data not shown). Whereas flowers held in glyphosate with 2 mM Phe plus 2 mM Tyr wilted at a similar rate to the controls (Fig. 3).
Glyphosate treatment of flowers delayed both the fading and wilting of mature flowers. Treat-ment of flowers with glyphosate in the presence of exogenous Phe and Tyr reversed the senescence-delaying effect of glyphosate when the concentra-Fig. 1. Changes in the colour of detached stage 7S.aurantiaca
flowers during senescence. Flowers were held in vase solutions of deionized water ( ) 1 mM Phe plus 1 mM Tyr (), 2 mM glyphosate () or 2 mM glyphosate and 1 mM Phe plus 1 mM Tyr ("). Values are the means of five replicates. L.S.D. (5%) between treatment means at certain days are shown by the vertical bars (d.f.=15).
J.R.Eason et al./Posthar6est Biology and Technology18 (2000) 81 – 84 84
Fig. 3. Wilting of detached stage 7S.aurantiacaflowers during senescence. The compressive strength was determined for flow-ers held in vase solutions of deionized water ( ) 2 mM glyphosate () or 2 mM glyphosate and 2 mM Phe plus 2 mM Tyr (). Values are the means of four replicates. L.S.D. (5%) between treatment means at certain days are shown by the vertical bars (d.f.=9).
is readily understandable in developing flowers, where metabolic processes (e.g. gametogenesis, pollen biogenesis, germination and tube growth) would require high rates of protein synthesis and high concentrations of phenylpropanoid precur-sors during development. It is now becoming equally clear that the same high rates of protein synthesis are required during flower senescence.
The data presented in this paper suggest that aromatic amino acids are important in the normal process of flower senescence. A reduction in the pool of aromatic amino acids, through glyphosate inhibition of enzymes in the shikimate pathway, may inhibit protein metabolism thereby altering the normal pattern of senescence.
References
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Killmer, J., Widholm, J., Slife, F., 1981. Reversal of glyphosate inhibition of carrot cell culture growth by gly-colytic intermediates and organic and amino acids. Plant Physiol. 68, 1299 – 1302.
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Weaver, L.M., Herrmann, K.M., 1997. Dynamics of the shiki-mate pathway in plants. Trends Plant Sci. 2, 346 – 351. tion of Phe and Tyr was ]2 mM. This suggests
that the effect glyphosate had on senescence was related to the metabolism of phenylalanine and tyrosine. The current data substantiates previous claims that the mode of action of glyphosate on plants is through its inhibitory effect on EPSP synthase and the subsequent reduction in the pool of aromatic amino acids (Killmer et al., 1981; Amrhein, 1986).
Most of the research on glyphosate has been undertaken to determine its mode of action as a herbicide. Glyphosate has been routinely used by the floriculture industry in devitalisation of cut roses and carnations to prevent bud outgrowth from the nodes. Here we report on the arrest of flower senescence by glyphosate. The shikimate pathway is primarily located in plastids (Gerhardt and Heldt, 1984; Amrhein, 1986). However, non-green tissues (e.g. flowers) contain the greatest amounts of mRNA for the inducible isozymes of the pathway (Weaver and Herrmann, 1997). A high demand for shikimate pathway intermediates
