Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

Heterogeneous changes in amino acid transport activity in E. coli lipid overproducing mutants

Mutants of E. coli that overproduce fatty acids or excrete phospholipids display heterogeneous changes in the transport rates for some but not all amino acids. Glutamic acid and proline are transported more rapidly than normal, asparagine and lysine are transported less rapidly. Transport rates for aspartic acid and glutamine are not altered. Only one of two kinetically distinct glutamate transport components has an elevated Vmax value. The findings suggest that individual amino acid transport catalysts are affected differently by the structural or metabolic changes evoked in these mutants.     

Amino Acid transport in germinating castor bean seedlings

During germination and early growth of the castor bean (Ricinus communis) nitrogenous constituents from the endosperm are transferred via the cotyledons to the growing embryo. Exudate collected from the cut hypocotyl of 4-day seedlings contained 120 millimolar soluble amino nitrogen and glutamine was the predominant amino acid present, comprising 35 to 40% of the total amino nitrogen. To determine the nature of nitrogen transfer, the endosperm and hypocotyl were removed and glutamine uptake by the excised cotyledons was investigated. Uptake was linear for at least 2 hours and the cotyledons actively accumulated glutamine against a concentration gradient. The uptake was sensitive to respiratory inhibitors and uncouplers and efflux of glutamine from the excised cotyledons was negligible. Transport was specific for the l-isomer. Other neutral amino acids were transported at similar rates to glutamine. Except for histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids. For glutamine transport, the K(m) was 11 to 12 millimolar and the V(max) was 60 to 70 micromoles per gram fresh weight per hour. Glutamine uptake was diminished in the presence of other amino acids and the extent of inhibition was greatest for those amino acids which were themselves rapidly transported into the cotyledons. The transport of amino acids, on a per seedling basis, was greatest for cotyledons from 4-to 6-day seedlings, when transfer of nitrogen from the endosperm is also maximal. It is concluded that the castor bean cotyledons are highly active absorptive organs transporting both sucrose and amino acids from the surrounding endosperm at high rates.     

Nitrogen assimilation in citrus trees

Assimilation of ¹⁵N-ammonium and ¹⁵N-nitrate was examined in 3-year-old satsuma mandarin (Citrus unshiu Marcovitch) trees. Experiments were designed to establish the time course of incorporation of nitrogen just taken up into amino compounds. In fine roots, absorbed ¹⁵N-ammonium was actively incorporated into glutamine and then into glutamic acid and asparagine. When feeding ¹⁵N-nitrate, glutamic acid and asparagine were actively synthesized, but glutamine synthesis was comparatively low as compared with that in ammonium feeding. In current leaves and fruits, a clear difference in the labelling patterns of amino acids was found between the ammonium and nitrate feedings. The amino acid most markedly labelled was asparagine in the ammonium feeding and glutamine in the nitrate feeding. Considering the most heavily labelled component in leaves and fruits, the main form of the nitrogen components transported upward in the xylem was discussed.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Photosynthetic assimilation of 14C into amino acids in potato (Solanum tuberosum) and asparagine in the tubers

Asparagine is the predominant free amino acid in potato tubers and the present study aimed to establish whether it is imported from the leaves or synthesised in situ. Free amino acid concentrations are important quality determinants for potato tubers because they react with reducing sugars at high temperatures in the Maillard reaction. This reaction produces melanoidin pigments and a host of aroma and flavour volatiles, but if free asparagine participates in the final stages, it results in the production of acrylamide, an undesirable contaminant. ¹⁴CO₂ was supplied to a leaf or leaves of potato plants (cv. Saturna) in the light and radioactivity incorporated into amino acids was determined in the leaves, stems, stolons and tubers. Radioactivity was found in free amino acids, including asparagine, in all tissues, but the amount incorporated in asparagine transported to the tubers and stolons was much less than that in glutamate, glutamine, serine and alanine. The study showed that free asparagine does not play an important role in the transport of nitrogen from leaf to tuber in potato, and that the high concentrations of free asparagine that accumulate in potato tubers arise from synthesis in situ. This indicates that genetic interventions to reduce free asparagine concentration in potato tubers will have to target asparagine metabolism in the tuber.     

Mechanism of amino Acid uptake by sugarcane suspension cells

The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.

Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge-compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site.

     

Post-uptake metabolism affects quantification of amino acid uptake

? The quantitative significance of amino acids to plant nutrition remains controversial. This experiment determined whether post-uptake metabolism and root to shoot export differ between glycine and glutamine, and examined implications for estimation of amino acid uptake. ? Field soil containing a Eucalyptus pauciflora seedling was injected with uniformly (13)C- and (15)N-labelled glycine or glutamine. I quantified (15)N and (13)C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of (12)CO(2) and (13)CO(2) from leaves and soil. ? 60-360 min after addition of amino acid, intact molecules of U-(13)C,(15)N glutamine were < 5% of (15)N excess in roots, whereas U-(13)C,(15)N glycine was 30-100% of (15)N excess in roots. Intact molecules of glutamine, but not glycine, were exported from roots to shoots. ? Post-uptake metabolism and transport complicate interpretation of isotope labelling such that root and shoot contents of intact amino acid, (13)C and (15)N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, (13)C and (15)N by determining the turnover of amino acids within roots. Alternatively, post-uptake metabolism and transport could be minimized by harvesting plants within minutes of isotope addition.     

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.     

Phloem Loading and Metabolism of Xylem-Borne Amino Compounds in Fruiting Shoots of a Legume

Cut, fruiting shoots of Lupinus albus L. supplied with ¹⁴C-and ¹⁵N-labelled L-asparagine, L-glutamine, L-aspartic acid,or L-glutamic acid through the transpiration stream readilytransferred the labelled carbon and nitrogen of each compoundto pods and seeds of fruits. A time course of labelling of phloemsap collected from petioles and fruit tips following feedingof labelled asparagine indicated that xylem to phloem exchangein leaflets was an immediate and effective route of transferof the amide to fruits and that this and the loading onto phloemof additional asparagine from unlabelled pools of the amidein stems furnished a major source of the nitrogen for fruitfilling. Xylem to phloem exchange of nitrogen was accomplishedin different ways for each amino acid. The amide nitrogen ofasparagine was transferred mainly in the unmetabolized compound,the nitrogen of aspartate and glutamate largely in a wide rangeof amino acids synthesized in the leaf, and the amide nitrogenof glutamine was transferred in a manner intermediate betweenthese extremes. Glutamine and asparagine were the principalphloem solutes labelled with nitrogen from any of the suppliedcompounds, but the photosynthetically produced amino acids,glutamate, aspartate, serine, alanine, and valine also became¹⁵N-labelled in phloem. The main pathway for glutamine synthesisin vegetative parts of the shoot appeared to be by amidationof glutamate, but asparagine was not considered to be derivedsimilarly from aspartate. Leaflets metabolized glutamine morereadily than asparagine, but in each case the amide nitrogenwas used for synthesis of a variety of amino acids and the carbonwas recovered largely in non-amino compounds.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.     

Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells

Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine’s effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. IEC-18; NIH/3T3; glutaminase; 6-diazo-5-oxo-L-norleucine; glutathione     

Gluconeogenesis from amino acids in germinating castor bean endosperm and its role in transport to the embryo

During germination of the castor bean all of the contents of the endosperm are ultimately transported to the embryo through the cotyledon or respired. A net loss of nitrogen from the endosperm begins about the fourth day, i.e. at the time when embryo growth and fat breakdown are also beginning. Amino acid analysis of the exudate from the cotyledons, still enclosed in the endosperm, showed that the amounts of aspartate, glutamate, glycine, and alanine were very low and that glutamine made up 40% of the amino acids in the exudate.

Amino acids labeled with ¹⁴C were applied to intact excised endosperms to follow utilization. Aspartate, glutamate, alanine, glycine, serine, and leucine were converted to sugar to varying extents. Proline, arginine, valine, and phenylalanine were not appreciably converted to sugars. Proline and glutamate were converted to glutamine. When ¹⁴C-glutamate, aspartate, and alanine were added to the outer endosperm of intact seedlings, only sugars and glutamine contained appreciable label in the exudate. When ¹⁴C-valine was added, it was virtually the only labeled compound in the exudate.

The results show that amino acids which on deamination can give rise to intermediates in the pathway of conversion of fat to sucrose are largely converted to sucrose and the nitrogen transported as glutamine. Other amino acids released from the endosperm protein are transported intact into the seedling axis. Some carbon from the gluconeogenic amino acids is also transported as glutamine.

     

Characterization of nitrogen relationships between Sorghum bicolor and the root-hemiparasitic angiosperm Striga hermonthica (Del.) Benth. using K15 NO3 as isotopic tracer

The role of the host in the nitrogen nutrition of Striga hermonthica (Del.) Benth. (Scrophulariaceae) parasitic on Sorghum bicolor cv. SH4 Arval has been investigated using (15)N-nitrate as the tracer. It is shown that, when nitrate is absorbed only by the roots of the host plant, a rapid transfer of nitrogen to the parasite can be detected. The xylem sap of S. hermonthica contained approximately equal amounts of nitrate and amino acids, mostly glutamine and asparagine. Infection altered the free amino acid profile of the host tissues, leading notably to a large increase in asparagine and a decrease in glutamine. The haustoria of S. hermonthica, although rich in nitrate, showed a low concentration of free amino acids, particularly lacking in asparagine and glutamine. The roots of S. hermonthica, in contrast, were rich in both asparagine and glutamine while, in the shoots, asparagine constituted 80% of the total FAA pool. Asparagine was also found to be the primary (15)N-enriched amino acid in the shoots of S. hermonthica while, interestingly, it was glutamate that was most strongly enriched in the roots. It is concluded that nitrogen nutrition in S. hermonthica is based on a supply of both nitrate and amino acids from the host. This implies a non-specific transfer in the transpiration stream. Nitrate reduction probably occurs mainly in the leaves of the parasite. Assimilation also occurs in S. hermonthica and excess nitrogen is stored as the non-toxic nitrogen-rich compound, asparagine. This specific trait of nitrogen metabolism of the parasite is discussed in relation to the effect of nitrogen fertilization on reducing infestation.     

Light-activated amino acid transport in Halobacterium halobium envelope vesicles.

Vesicles prepared from Halobacterium halobium cell envelopes accumulate amino acids in response to light-induced electrical and chemical gradients. Nineteen of 20 commonly occurring amino acids have been shown to be actively accumulated by these vesicles in response to illumination or in response to an artificially created Na-gradient. Sodium-activated amino acid transport for 18 of these amino acids has been shown to occur in direct response to the protonmotive force generated. Glutamate is transported only in response to a sodium gradient. Michaelis constants for the uptake of these amino acids are close or identical whether the amino acids are accumulated in response to a sodium gradient or a protonmotive force (i.e., electrical gradient). On the basis of shared common carriers the transport systems can be divided into eight classes, each responsible for the transport of one or several amino acids, i.e., arginine, lysine, histidine; asparagine, glutamine; alanine, glycine, threonine, serine; leucine, valine, isoleucine, methionine; phenylalanine, tyrosine, tryptophan; aspartate; glutamate; proline. Available evidence suggests that these carriers are symmetrical in that amino acids can be transported equally well in both directions across the vesicle membranes. A tentative working model to account for these observations is presented.     

A comparison of amide and ureide nitrogen sources in tissue culture of tree legume Prosopis alba clone B2 V50

The effects of medium nitrogen sources on the recalcitrant nature of Prosopis alba clone B2V50 in tissue culture were compared involving shoot development using axillary bud explants from 2 to 4-year-old greenhouse-grown trees. A significant difference (P<0.05) was found between the amino acids aspartic acid and glutamic acid and their corresponding amide-containing compounds asparagine and glutamine. A comparison between amide and ureide nitrogen sources showed that allantoin, a ureide, was an acceptable replacement for asparagine or glutamine. Allantoin, asparagine, and glutamine could be used as the sole nitrogen sources. Allantoin at a concentration of 20 mM was adopted for use in future research. Although shoots were consistently induced, all explants showed complete shoot-tip necrosis after 12 weeks of in vitro culture.     

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [³H]aspartic acid and [¹⁴C]asparagine. The aspartic acid pool turned over rapidly with a half-time of <30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as ～25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material. © 1994 Wiley-Liss, Inc.     

INFLUENCE OF AMMONIUM AND NITRATE ON THE PROTEIN- AND FREE AMINO ACIDS IN SHOOTS OF WHEAT SEEDLINGS

Weissman , Gerard S. (Rutgers U., Camden, N. J.) Influence of ammonium and nitrate on the protein- and amino acids in shoots of wheat seedlings. Amer. Jour. Bot. 46(5): 339–346. 1959.—Total and protein nitrogen per shoot of wheat seedlings grown with endosperm attached increased at a steady rate during a 96-hr. growth period, and protein nitrogen, as a percentage of total nitrogen, remained constant at about 53%. Total and protein nitrogen concentration was greatest for 24-hr. shoots and declined as the shoots became older. Total and protein nitrogen were determined in 96-hr. shoots of seedlings grown with endosperm attached but also supplied with ammonium, nitrate, or both in the culture solution. Total nitrogen was greatest in shoots supplied with ammonium, but only 38% was in the form of protein. Maximum protein synthesis occurred in shoots grown in both ammonium and nitrate and protein nitrogen as a percentage of total nitrogen approximated that achieved in shoots lacking nitrogen in the culture solution. The protein amino acid composition of 48-, 72-, and 96-hr. shoots was very similar but differed from 24-hr. shoots which contained higher percentages of arginine and lysine and lower percentages of alanine and threonine. This may be correlated with the higher proportion of meristematic cells in 24-hr. shoots. The protein amino acids in shoots grown with ammonium resembled that of shoots lacking nitrogen in the culture solution, but nitrate shoot protein contained a higher percentage of arginine and a lower percentage of lysine. Nitrate may stimulate the formation of enzymes, possibly of a nitrate-reducing system, with high arginine- low lysine content. Free asparagine and glutamine were both at a maximum in ammonium shoots and at a minimum in nitrate shoots, but asparagine predominated in shoots supplied with ammonium while glutamine was greatest in nitrate shoots. Aspartic acid, asparagine, and glutamine appeared to have ammonia-storage functions, but glutamic acid appeared to be primarily concerned with protein synthesis. Amino acid accumulation was greatest in shoots supplied with both ammonium and nitrate. Protein synthesis in these appeared to be limited by inadequate concentrations of glutamic acid and proline. A hypothesis is proposed in explanation of the high glutamic acid concentration in shoots provided with ammonium and nitrate.