Produced by the phytopathogenic fungus Alternaria tenuis, it induces chlorosis in many plants, including lettuce, potato, cucumber, and spinach, but has little or no effect on others such as radish, tobacco, and corn (4, 5). The chlorosis results from a selective disruption of chloroplast function in that the amounts of lipids (6) and proteins (7) specific to chloroplasts are reduced and the ultrastructural alterations found are confined to the chloroplast (8). The sharp demarcation between sensitive and insensitive species suggested that chloroplasts from sensitive species might possess a specific receptor site for tentoxin. Because preliminary studies have shown that tentoxin inhibits phosphorylating electron transport in lettuce chloroplasts (9) and chloroplast coupling factor 1 (CF1) is directly associated with photophosphorylation (10), we investigated it as a potential receptor molecule for tentoxin. This paper presents evidence that CF1 from lettuce, a tentoxin-sensitive species, has a single binding site for tentoxin and that when tentoxin occupies this site, both CF1 ATPase and phosphorylating electron transport are inactivated. In contrast, CF1 from radish, an insensitive species, has a lower affinity for tentoxin than does lettuce CF1; tentoxin does not inhibit radish ATPase and only slightly inhibits photophosphorylation.MATERIALS AND METHODS Chloroplasts were prepared from leaves of lettuce (cv. Romaine) and spinach purchased locally and from seedlings of radish (cv. Comet) and other species grown in controlled environment chambers. One hundred grams of selected leaves were blended for 10 sec at 40 in 250 ml of a 0.4 M sucrose 0.05 M N-[tris(hydroxymethyl)methyl]glycine (Tricine) buffer, pH 8.0, (ST). The homogenate was filtered through eight layers of cheesecloth, and the chloroplasts were collected by centrifugation (1000 X g for 10 min). After washing in 40 ml of ST and an additional centrifugation, the chloroplast pellet was suspended in 10 ml of ST and adjusted to 1 mg of chlorophyll per ml (11).Electron transport was measured with an oxygen electrode using methods modified from those of Arntzen (9). Chloroplasts (35 ,g of chlorophyll) in ST were added to 3 ml of stirred reaction mixture at 280 followed by the addition of tentoxin. Next, the electrode was inserted during a brief cessation of stirring. Two minutes after the addition of tentoxin, the reaction chamber was illuminated with a tungsten lamp (15 klm/m2). The course of oxygen uptake was followed during the subsequent 1 min. Basal rates were measured in the presence of 3 mM ADP; complete rates were determined in the added presence of 5 mM NaH2PO4. Only preparations having a complete rate more than twice the basal rate were used.Coupling factor 1 was prepared from chloroplasts by the methods of Lien and Racker (12). Lettuce CF1 was judged to be pure by gel electrophoresis (13, 14) after chromatography on DEAE-Sephadex A-50 using a linear (NH4)2SO4 gradient (Step 3 of ref. 12). Radish CF1 of comparable purity was obtained after initial co...
Tabtoxinine-f-lactam, a hydrolytic product of tabtoxin produced by Pseudoonas syringae pv. tabaci, apparently inactvates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 minhmmolar tabtoxinne-/-lactam in the presence of 10 m_mlar glutamate. A method for the purification of glutamine synthetase from dried peas is presented which gives a 30% yield with a 2,000-fold Increase in specific activity. A method for obtaining highly puied tabtoxinine-f8-lactam and tabtoxin in good yields Is also presented. The authenticity and purity of tabtoxinine*lactam and tabtoxin were verified by chromatography, biological activity, and 1H and 13C nuclear magnetic resonance spectroscopy.The phytopathogenic bacterium Pseudomonas syringae pv. tabaci and certain other closely related pathovars produce a toxin which induces chlorosis in plants (5,11,12,16). This toxin, called tabtoxin, is a dipeptide composed of either threonine or serine and the novelf,-lactam-containing amino acid, tabtoxinine-,8-lactam (2-amino-4-[3-hydroxy-2-oxo-azacyclobutan-3-yl]-butanoic acid).Originally, it was hypothesized that chlorosis was caused by a rapid buildup of ammonium, resulting from the inhibition of GS' (EC 6.3.1.2) by tabtoxin. Semipurified tabtoxin did, in fact, inhibit the activity of crude GS from pea (14). This hypothesis was also supported by the following observations: (a) MSO, which was already known to inhibit GS, also caused chlorosis and ammonium accumulation (7, 10); (b) tabtoxin-and MSO-induced chlorosis and ammonium accumulation are light-dependent reactions (3) which can be inhibited by dichlorodimethyl phenylurea (Durbin, unpublished); and (c) glutamine negated the inhibitory effects of both MSO and tabtoxin in several biological systems (2). Preliminary studies using purified materials failed to show inhibition of GS by tabtoxin (19). This has been clarified by the present study and the recent observation that unidentified peptidases in tobacco will hydrolyze tabtoxin to yield tabtoxinine-,flactam (19). Inasmuch as this compound can also cause chlorosis and, more important, appeared to inhibit GS, we postulated that chlorosis development via an inhibition of this enzyme depended upon the generation of tabtoxinine-,B-lactam.This report establishes that purified tabtoxinine-fl-lactam, but not tabtoxin, inhibits purified GS from pea by apparently irreversibly inactivating the enzyme. A simple, rapid procedure for the purification of tabtoxin and tabtoxinine-f,-lactam is also reported.'Abbreviations: GS, glutamine synthetase; MSO, L-methionine-S-sulfoximine; NMR, nuclear magnetic resonance. MATERIALS AND METHODSEnzyme Purification. Commercially obtained, dried split peas (1 kg) were milled to a fine powder and soaked 24 h at 4°C in 2 L 20 mm imidazole (pH 7.5), 10 mM MgC12, and 1 mM ,B-mercaptoethanol. The crude enzyme preparation was clarified by centrifugation (14,000g for 30 min), and a fraction containing the enzyme activity was precipitated by adjusting the pH to ...
Summary. Tabtoxins, dipeptides that induce chlorosis in plants, are produced by several closely related phytopathogenic Pseudomonas spp. Culture filtrates from these bacteria and extracts prepared from leaves of their hosts contain multiple enzymes that hydrolyze the peptide bond of the toxins.The phytopathogenic bacterium Pseudomonas tabaei (Wolf and Foster) Stevens and several closely related species produce toxins that cause chlorosis in higher plants 1 and inhibit the growth of Escherichia coli (Migula) Castellani and Chalmers (Durbin, unpublished data). Called tabtoxins, they are dipeptides containing tabtoxinine-fl-lactam [2-amino-4-(3-hydroxy-2-oxo-azocyclobutan-3-yl)-butanoic acid] linked to either threonine or serine 2'3. Originally it was postulated that these toxins acted as antimetabolites of methionine, partially because of the similarity in activity of the toxins to methionine sulfoximine (MSO), which at that time was also thought to affect methionine metabolism 4. Later investigations showed that although methionine would compete with MSO for passage across the plasmalemma 5, its actual site of action is glutamine synthetase (GS) 6. Initial experiments with crude preparations of the tabtoxins and GS supported the idea that the tabtoxins also inhibited GS 7. However, in further studies using purified components and a sensitive assay s, no inhibition of GS could be demonstrated (Durbin and Uchytil, unpublished data). More recently, free tabtoxinine-fl-lactam has been found in culture filtrates of P. tabaci 9. Most interestingly, as with the tabtoxins, this compound causes chlorosis, and, as with the toxins, the reaction is light dependent and there is an accumulation of ammonia in chlorotic tissue ~~ Furthermore, chlorosis is always evident earlier than with the tabtoxins. These findings support the hypothesis that tabtoxinine-fl-lactam could playa key role in vivo in chlorosis production. If so, the peptide bond of the tabtoxins would have to be cleaved. The present studies provide evidence that both plants and Pseudomonas spp. have enzymes with peptidase activity capable of hydrolyzing the tabtoxins, and discusses the possible significance of these findings to the mechanism of action.Materials and methods. Peptidase activity of bacterial culture filtrates was tested by growing 5 isolates of P. tabaci and 3 isolates of P. coronafaciens (Elliott) Stevens for 5 days with agitation in Woolley's medium ~1 at 26 ~ Cells were removed by centrifugation (12,000x g for 15 rain), sodium azide added to give a final concentration of 0.05%, and the filtrate sterilized (Millipore, 0.45 pro). A portion of the filtrate was maintained at 4 ~ and the remainder incubated at 37 ~ for 12 h. Additional controls consisted of adding 0.01% diethylpyrocarbonate (v/v), a histidine-modifying reagent ~2, to the filtrate incubated at 37 ~ or adjustment of filtrate from pH 7.3 to 2 or 10. After incubation, aliquots from each treatment with and without the addition of the tabtoxins and their hydrolytic products 2 were chromatogr...
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