Inhibition by p-Fluorophenylalanine of Protein Synthesis and of Ion Transport Across the Roots in Barley Seedlings

Azetidine 2-carboxylic acid (AZ) was used as an analog of proline to investigate further the relationship between protein synthesis and ion transport. AZ does not inhibit protein assembly, but the proteins formed are ineffective as enzymes. At relatively low concentrations (50 5LM) AZ was a potent inhibitor of release of ions to the xylem of excised roots of barley (Hordeum vulgare L.) and intact plants. Uptake to the root was also inhibited but to a lesser degree. A procedure was introduced for estimating unidirectional fluxes from measurements of net tracer uptake, net transport to the xylem, and net efflux from the roots. It was shown that inhibition of release to the xylem was not caused by reduction in influx at the plasmalemma or to stimulation of influx to the vacuoles. It was suggested that AZ was acting on the process of release from symplast to the xylem. The action of AZ is compared with similar effects on ion transport produced by p-fluorophenylalanine, cycloheximide, and abscisic acid.

Section: Reid (Personal Communicationmentioning

confidence: 99%

mentioning

confidence: 93%

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Effect of Azetidine 2-Carboxylic Acid on Ion Uptake and Ion Release to the Xylem of Excised Barley Roots

Pitman¹,

Wildes²,

Schaefer³

et al. 1977

“…The relatively long time (at least 6 h) for the stimulus in P uptake to be initiated by ammonium and its persistence upon transfer to N-free media (20) suggest a possible involvement of protein synthesis in its induction. Addition of the analogs of phenylalanine (p-FPA) and proline (AZ) to root tissue has been shown to result in synthesis of ineffective protein (15,17,18) and, over longer periods, in reduced protein synthesis (15). Accordingly experiments involving presence of these analogs during the pretreatment period were conducted with genotype DeKalb XL-45 to determine if induction of the stimulus would be eliminated when synthesis of functional protein was impaired.…”

Section: Methodsmentioning

confidence: 99%

Nitrogen Enhancement of Phosphate Transport in Roots of Zea mays L

Smith¹,

Jackson²

1987

Exposure to 1 millimolar ammonium pretreatments increased V., for phosphorous uptake in dark-grown decapitated maize seedlings without a statistically measurable change in K.. Sulfate uptake also was stimulated. The stimulation in phosphorous uptake due to ammonium pretreatment was greater in seedlings grown without phosphorous than in those grown with 25 micromolar phosphorous. The stimulus was not expressed unless the entire root system was pretreated with ammonium, and pretreatment of a part of the root system inhibited phosphorous uptake by the remaining part unless it also had been pretreated. Pretreatment with the amino acid analogs p-fluoro-DL-phenylalanine and L-azetidine-2-carboxylic acid (AZ) restricted phosphorous uptake in seedlings that were pretreated with ammonium and in those that were not, but the effect ofammonium pretreatment was not completely eliminated by the analogs. In general, translocation of the entering phosphorous was affected similarly to uptake by experimental treatments. Enhanced translocation, however, was not sufficient to account quantitatively for the increase in uptake, and an increased uptake was still evident when translocation was completely prevented by 50 micromolar AZ pretreatment.Many agronomic studies on the banding of nitrogen (N) and phosphorous (P) fertilizers have shown that N increases the absorption offertilizer P. While there are a number of soil related factors and interactions between the plant and soil that contribute to this effect (7, 16), it is recognized that the phenomenon is partially attributable to physiological reactions of the plant itself (16). Among the earliest studies to arrive at this conclusion was that of Cole et al. (5) who eliminated soil related factors and found up to 3-fold increases in P uptake by N pretreated maize plants grown in solution culture. They suggested a link between synthesis of N intermediates such as glutamine and the energy supply needed for anion uptake.Other studies sought to explain the phenomenon by suggesting that N increased the rate of transfer of P across the root symplast to the xylem. Leonce and Miller (13) hypothesized that ammonium may increase the rate at which a phosphorous-carrier complex released P into the xylem. Later observations that N pretreatment stimulated P translocation to the tops relatively more than P uptake, led to the suggestion that the N effect was exerted on two quite distinct P transport systems: the initial uptake across the plasmalemma and the subsequent lateral movement of P to the xylem (21, 22). It was postulated that some N intermediates, presumably synthesized during the pretreatment phase, influenced these two processes.We have shown that the stimulation of P uptake could be induced in decapitated maize seedlings by pretreatments with either nitrate or ammonium. In presence of ammonium, the stimulus took 6 to 8 h to develop and did not decay rapidly (20). Development of the stimulus therefore did not require participation by shoot-related processes. The present paper d...

“…For example, cycloheximide has been reported to stimulate respiration and inhibit chloride uptake by beet root discs within 1 h of its application (8). Because of this lack of specificity, we have reexamined the effect of impaired protein synthesis on nitrate uptake and assimilation, using FPA,3 an analog of phenylalanine, to produce ineffective protein (18,21). Potassium fluxes were also quantified, since this cation is the common counterion associated with nitrate uptake and translocation.…”

mentioning

confidence: 99%

p-Fluorophenylalanine-Induced Restriction of Ion Uptake and Assimilation by Maize Roots

Morgan¹,

Volk²,

Jackson³

1985

ABSTRACIRoots of decapitated maize seedlings (Zea mays L.) were exposed for 12 hours to 1.0 millimolar KNO3 (98.5 atom per cent 15N) in the presence and absence (control) of 0.1 millimolar p-fluorophenylalanine (FPA), an analog of the amino acid phenylalanine. FPA decreased nitrate uptake but had little effect on potassium uptake. In contrast, accumulation of both ions in the xylem exudate was greatly restricted. Utilization of nitrate by higher plants involves the basic processes of uptake, reduction and translocation. Nitrate reductase, the enzyme which catalyzes the rate-limiting step in nitrate assimilation, requires continued protein synthesis to maintain its activity (10,11,16,23,24). There is also evidence that uptake and translocation of nitrate are dependent on protein synthesis (9,19,26). This latter evidence, however, is based primarily on studies with inhibitors of RNA and protein synthesis, some of which are known to affect cellular processes other than protein synthesis (3, 7, 8). For example, cycloheximide has been reported to stimulate respiration and inhibit chloride uptake by beet root discs within 1 h of its application (8). Because of this lack of specificity, we have reexamined the effect of impaired protein synthesis on nitrate uptake and assimilation, using FPA,3 an analog of phenylalanine, to produce ineffective protein (18,21). Potassium fluxes were also quantified, since this cation is the common counterion associated with nitrate uptake and translocation.'Supported by National Science Foundation Grant PCM 81-18661. MATERIALS AND METHODSGrowth of Maize Seedlings. DeKalb XL-45 maize seeds (Zea mays L.) were germinated on paper towels moistened with 0.1 mM CaSO4 in an incubator maintained in darkness at 30°C and 98% RH. After 3 d, all roots except the primary roots were excised, and the seedlings were assembled into cultures of five seedlings each. The cultures were placed into aerated, nitrogenfree nutrient solution and returned to the incubator. The solution (pH 6.0) contained 0.05 mm K2S04, 0.4 mM KH2PO4, 0.25 mM MgSO4, 1.0 mm CaSO4, trace elements at 0.4 Hoagland concentration (12) and FeEDTA at 1.0 mg Fe 1-'. On the 4th d, the shoots were excised below the first node, and exudate collectors, small plastic pipet tips, were sealed to the mesocotyls with silicone grease. The cultures were placed in fresh, nitrogen-free nutrient solution and returned to the incubator. The experiment was run 20 h later, when the decapitated seedlings were 5-d old.Experimental Procedure. Just prior to the experiment, exudate was removed from the collectors, and the roots were rinsed thoroughly in 1.0 mm CaSO4 (30°C). The roots of each culture were then exposed to 250 ml of uptake solution containing 1.0 mM K'5NO3 (98.5 A % '5N) and 0 (control) or 0.1 mm FPA. Otherwise, the composition of the uptake solution was identical to that of the nitrogen-free, nutrient solution. During the 12-h exposure period, the uptake solutions (pH 6.0) were maintained at 30°C, aerated continuously, and replaced with fresh solu...