Determination of Ammonium, Amide, Nitrite, and Nitrate Nitrogen in Plant Extracts

Mobilization of N from leaves of barley (Hordeum vulgare L.) during water stress, and the role of proline as a mobilized species, were examined in plants at the three-leaf stage. The plants responded to water stress by withdrawing about 25% of the total reduced N from the leaf blades via phloem translocation. Most of this N loss was during the first 2 days while translocation of 'C-photosynthate out of the stressed blade still remained active. Free proline accumulation in the blade was initially slow, and became more rapid during the 2nd day of stress. Although a major free amino acid, proline accounted for only about 5% of the total N (soluble + insoluble) retained in severely stressed blades. When the translocation pathway in water-stressed leaves was interrupted just below the blade by a heat girdle, a cold jacket, or by blade excision, N loss from the blade was prevented and proline began to accumulate rapidly on 1st day of stress. Little free proline accumulated in the blades until after the ability to translocate was lost. Proline was, however, probably not a major species of N translocated during stress, because proline N accumulation in heatgirdled stressed leaves was five times slower than the rate of total N export from intact blades.The onset of water deficit alters the over-all N budget of crops through effects on both N transport and N assimilation. Nitrate delivery via the xylem to the shoot is depressed (17), and N03 reduction in the leaves declines (12,17). Although phloem transport of assimilated N to N sinks is eventually reduced (4, 25), it is probable that phloem transport is sufficiently resistant to water stress to permit appreciable salvage of both N and carbon from wilted leaves that are dying as a consequence of desiccation (3,6,25).Within the leaves of many plants subjected to moderate or severe water stress, one striking change in N metabolism is the accumulation of free proline as a result of net de novo synthesis from glutamic acid (e.g. 1, 2). It has been proposed that this accumulation is a metabolic adaptation which confers survival value, perhaps acting as a (phloem-mobile) reserve of N accessible for use upon stress relief (e.g. 1,18,21). This possibility cannot be evaluated without knowing: (a) how much N is exported from stressed leaves to other parts of the plant; and (b) whether proline accumulation and export are significant terms in the total N budget of a leaf during stress. Much of the published work on proline accumulation during water stress has been with detached leaves, which clearly cannot export N. Proline accumulation has also been investigated in intact plants. In Plants were grown in Perlite in plastic pots (7 x 12 cm) for 17 to 19 days after sowing (to the three-leaf stage) in a growth chamber under a regime of 16-h days, day/night temperature 22/ 16 C, day/night RH 70/85%, total irradiance 3.8 mw cm-2 (80%o from fluorescent lamps, 20o from incandescent lamps), and were watered on alternate days with half-strength Hoagland solution.Prior to the exper...

Section: Methodsmentioning

confidence: 99%

Proline Accumulation in Water-stressed Barley Leaves in Relation to Translocation and the Nitrogen Budget

Tully¹,

Hanson²,

Nelsen³

1979

“…After the cotyledons were removed, the NH4+ from 10 ml of media was distilled from a sodium borate buffer and trapped in a boric acid buffer (21 Double cross-polarization "N NMR spectra were obtained using matched spin-lock transfers first from 'H to "N and then from N to 13C. If the "3C-rf field is on resonance and its amplitude satisfies a carbon-nitrogen Hartmann-Hahn condition, a spin-lock transfer from "N to 13C drains polarization from "N. A direct difference experiment between single and double cross-polarization procedures then results in the accumulation of a DCPMAS difference "N signal arising exclusively from the "N directly bonded to 13C.…”

Section: Fragmentmentioning

confidence: 99%

¹⁵N and ¹³C NMR Determination of Allantoin Metabolism in Developing Soybean Cotyledons

Coker¹,

Schaefer²

1985

The metabolism of allantoin by immature cotyledons of soybean (Glycine max L. cv Elf) grown in culture was investigated using solid state 13C and '5N nuclear magnetic resonance. All of the nitrogens of allantoin were incorporated into protein in a manner similar to that of each other and to the amide nitrogen of glutamine. The C-2 of allantoin was not incorporated into cellular material; presumably it was lost as CO2. About 50% of the C-5 of allantoin was incorporated into cellular material as a methylene carbon; the other 50% was presumably also lost as CO2. The 13C-'5N bonds of 15-'3C;1-'5N1 and 12-'3C;1,3-'5Nlallantoin were broken prior to the incorporation of the nitrogens into protein. These data are consistent with allantoin's degradation to two molecules of urea and one two-carbon fragment. Cotyledons grown on allantoin as a source of nitrogen accumulated 21% of the nitrogen of cotyledons grown on glutamine. Only 50% of the nitrogen of the degraded allantoin was incorporated into the cotyledon as organic nitrogen; the other 50% was recovered as NI-L in the media in which the cotyledons had been grown. The latter results suggests that the lower accumulation of nitrogen by cotyledons grown on allantoin was in part due to failure to assimilate NH4W produced from allantoin. The seed coats had a higher activity of glutamine synthetase and a higher rate of allantoin degradation than cotyledons indicating that seed coats play an important role in the assimilation and degradation of allantoin.

“…The tubes were warmed to 55 to 65 C and held at this temperature for 30s; 0.25 ml CHC13 and 0.4 ml H20 were then added, and the tubes were shaken and centrifuged. The aqueous phase (volume about I ml) was drawn off, and samples were taken for scintillation counting and for determination of13NH3 after vacuum distillation (17). For identification of labeled organic products, the aqueous fraction was evaporated in vacuo at 55 C to a small volume ,ld).…”

mentioning

confidence: 99%

Amino Acids Translocated from Turgid and Water-stressed Barley Leaves

Hanson¹,

Tully²

1979

Movement of labeled amino acids from leaf blades to sheaths was followed after supping "NH3 gas, '4CO2, or '4C-amino-acids to attached blades of barley plants. Blades The phloem exudation studies in the preceding paper (15) indicated that glutamine and glutamate are two of the major amino acids in phloem sap of both unstressed and water-stressed barley plants, and that proline is a minor phloem amino acid in stressed plants. Further tests of the importance of glutamine, glutamate, and proline in phloem transport of N were made using two radioactive tracer techniques.First, leaf blades of intact plants were exposed to gaseous NH3