Total nitrogen and free amino acids in Morus alba stems from autumn through spring

During leaf senescence and abscission, total nitrogen in leaves of mulberry ( Morus alba L. ev. Shin-ichinose) declined substantially whereas total nitrogen in buds, bark and stem wood increased markedly, suggesting translocation of nitrogen from senescent leaves in the autumn. After leaf abscission the winter buds and stems remained almost unchanged with respect to fresh and dry weight and total nitrogen until bud break in spring. In burst buds these parameters then increased drastically during the new growth while they decreased markedly in stems. Free arginine in the stem bark accumulated in parallel with the accumulation of total nitrogen in buds and stems in the autumn. Accumulation of proline in the wood, bark and buds also started in October but continued even after leaf-fall, increasing until mid-January (wood), mid-February (bark) and the new growth (buds). Prior to and in the early stage of bud break, proline in bark and wood decreased significantly and arginine in stem bark decreased slightly. Simultaneously, proline and arginine in the dormancy-releasing buds and asparagine, aspartic acid and glutamic acid in the buds and stems increased appreciably, suggesting that this increase in free amino acids was mainly derived from free amino acids (proline and arginine) stored in stems. The resulting marked decrease in total nitrogen and the drastic increase in asparagine in the stems and sprouting buds/new shoots were primarily due to a breakdown of protein stored in stems.     

Free amino acids and total nitrogen during shoot development in Scots pine seedlings

The concentration of free amino acids and total nitrogen was studied in needles, stems and roots of seedlings of Pinus sylvestris L. for five weeks during the second growth period ("summer"). In one group of seedlings the source/sink relation was disturbed through removal of the terminal buds. The seedlings were cultivated in artificial year-cycles in a climate chamber.
Total nitrogen increased in needles and sterns of intact seedlings in the beginning of the "summer" and decreased during shoot growth. In seedlings, from which the buds had been removed, nitrogen remained at high levels in the primary needles and accumulated in steins and roots. The results are consistent with utilization of nitrogen in older needles and in the stem during shoot elongation.
The pool of free amino acids increased in the beginning of the "summer" and decreased after bud break in primary needles, stems and roots. Arginine and glutamine, in the roots also asparagine, were the dominating amino acids (amides included). Together, these compounds (plus glutamate and aspartate) contributed about 90% of the nitrogen in the amino acid pool in all organs. In primary needles and in the stem, arginine predominated at the end of hardening (75–85% of the amino acid nitrogen). Free amino acids contributed at most ca 10% of the total nitrogen in primary needles, where the ratio of free amino acid nitrogen: total nitrogen was highest at the end of dormancy and in the early "summer". Free amino acids accumulated after bud removal in primary needles and especially in stems and roots. Glutamine became relatively more dominant than arginine in the different organs.
The observations are consistent with the role of arginine and glutamine for storage and transport of nitrogen in conifers. Because of the low concentrations of amino acid nitrogen in the primary needles, arginine is not considered a major nitrogen reserve in needles of Scots pine seedlings.     

Export of amino acids to the phloem in relation to N supply in wheat

The effect of different N supply on amino acid export to the phloem was studied in young plants of wheat (Triticum aestivum L. cv. Klein Chamaco), using the exudation in EDTA technique. Plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different NO₃^? concentrations. When plants were grown for 15 days with nutrient solutions containing 1.0, 3.0, 5.0, 10.0, 15.0 or 20.0 mM KNO₃, the exudation rate of sugars from the phloem was unaffected by N supply, but sugars accumulated in the leaf tissue when the N supply was limiting for growth. On the other hand, the rate of exudation of amino acids was proportional to the NO₃^? concentration in the nutrient solution. When the supply of N to plants grown for 15 days with 15.0 mM NO₃^? was interrupted, the exudation of sugars was again unaffected, but there was a fast decrease in the amount of amino acids exudated, and of the concentration of amino acids and nitrogen in the tissues. Also, when 10-day-old plants grown without N were supplied with 15.0 mM NO₃^?, there was a sharp increase in the exudation of amino acids, without changes in the amount of sugar exudated. The rate of exudation of amino acids to the phloem was independent of the concentration of free amino acids in the leaves in all three types of experiment. Asp was the most abundant amino acid in the leaf tissue, while Glu was the one most abundant in the phloem exudate. Asp and Ala were exported to the phloem at a rate lower than expected from their leaf tissue concentrations, indicating some discrimination. On the contrary, Glu showed a preferential export at low N supply. It is concluded that the rate of amino acid export from the leaf to the phloem is dependent on the N available to the plant. This N is used for synthesis of leaf protein when the supply is low, exported to the phloem when supply is adequate, and accumulated in the storage pool when supply is above plant demand.     

Changes in free amino acids and osmotic adjustment in leaves of water-stressed bean

Bean ( Phaseolus vulgaris L. cv. Topcrop) plants were raised in a growth chamber in small pots or flower boxes and kept at full water regime until the full development of primary leaves (14–16 days). Both potted and flower box-grown plants were subjected to a gradually increased water stress of about 60–70 kPa day⁻¹ leaf water potential (stressed plants) or full water regime (control). The water potential, osmotic potential and turgor pressure in freshly detached primary leaves and osmotic potential at zero turgor were calculated using pressure/volume curves. Changes in free amino acids and amides were also measured in parallel trials. Water relation parameters documented that in the stressed leaves there was moderate osmotic adjustment, which was more evident in the potted plants. If considered 0% ionised, the accumulation of free amino acids and amides (μmol g⁻¹ H₂O) accounts for a van't Hoff's value of about 10.2 kPa in the small pot-grown and 5.5 kPa in the box-grown plants. The values are twice as high if considered 100% ionised. Proline accumulation accounted for about 6.4% of the pool enlargement in the potted plants and 22.3% in the flower box plants.     

Influence of drought on the concentration and distribution of 2,4-diaminobutyric acid and other free amino acids in tissues of flatpea (Lathyrus sylvestris L.)

2,4-Diaminobutyric acid (A₂bu) may be responsible for the apparent toxicity of flatpea (Lathyrus sylvestris L.) forage to some livestock. To obtain information relative to environmental regulation of A₂bu, 3-month old flatpea plants, cv. “Lathco”, were subjected to water-deficit stress for 1, 2, and 4 weeks. A₂bu, the most abundant free amino acid in roots, stems, and leaves, increased nearly 100% in roots of stressed plants. Increases in the concentrations of asparagine (Asn), proline (Pro), and arginine (Arg) occurred in roots; Asn, Pro, and 4-aminobutyric acid (Abu) in stems; and Pro and homoserine (Hse) in leaves also occurred in response to drought stress. Proline was a minor constituent of the free amino acid pool, even under water-deficit stress. The distribution of A₂bu and Pro in the stressed plants (roots > stems > leaves) was the reverse of that in plants supplied with adequate water (roots < stems < leaves). As concentrations of Asn and Abu decreased from roots to leaves in control tissues, concentrations of Hse and A₂bu increased in roughly the same proportions. This observation suggests that Abu and Asn may be precursors of A₂bu and Hse, respectively. The increase in A₂bu levels in aerial parts of drought-stressed flatpea plants is probably not sufficient to lower the feed value of the forage.     

Nitrogen distribution in young Norway spruce (Picea abies) trees as affected by pedospheric nitrogen supply

In numerous locations in Europe spruce trees are exposed to high loads of nitrogen. The present study was performed to characterize the distribution of nitrogen compounds under these conditions. For this purpose Norway spruce ( Picea abies [L.] Karst.) trees were cultivated under close-to-natural conditions of a forest understory in soil from an apparently nitrogen-limited field site in the Black Forest either with, or without supplementation of nitrogen as ammonium nitrate. After 11 and 20 months, growth, total nitrogen contents of the biomass, and total soluble non-proteinogenic nitrogen compounds (TSNN, i.e. nitrate, ammonium, soluble proteinogenic and non-proteinogenic amino compounds) in needles, xylem sap and phloem exudate were analysed. After 20 months of growth, N-fertilization had slightly enhanced the biomass of current-, but not of 1-year-old shoots. At both harvests, total N-content of 1-year-old needles was increased by N-fertilization, whereas current-year needles were not significantly affected. By contrast, TSNN was elevated by N-fertilization in both current-year and 1-year-old needles. The increase in TSNN was mainly attributed to an accumulation of arginine. Xylem sap analysis showed that the increase in TSNN of the needles was a consequence of enhanced nitrogen assimilation of the roots rather than the shoot. Since also TSNN in phloem exudates was enhanced, it appears that N-fertilization elevates the cycling pool of amino compounds in young Norway spruce trees. However, this pool seems to be subject to metabolic interconversion, since mainly glutamine and aspartate are transported in the xylem from the roots to the shoot, but arginine accumulated in the needles and the phloem.     

Difficulties in location and acceptance of phloem sap combined with reduced concentration of phloem amino acids explain lowered performance of the aphid Rhopalosiphum padi on nitrogen deficient barley (Hordeum vulgare) seedlings

Effects of nitrogen deficiency in hydroponically grown barley seedlings (Hordeum vulgare L.) on the development and reproduction of the aphid Rhopalosiphum padi (L.) (Hemiptera: Aphididae) were investigated.Plant growth was significantly reduced in seedlings grown without nitrogen. Aphid intrinsic rate of increase (r _m) was also significantly lower on these plants compared with that on plants grown with 8 mol m^–3 nitrogen. Phloem sap was collected from seedling stems by aphid stylectomy and amino acids quantified by HPLC. There was a significant reduction in the concentration of non-essential amino acids as a group, but not of essential amino acids. Electrical penetration graphs (EPG) indicated that aphids reached the phloem more quickly and fed for longer on plants grown with nitrogen. This is the first reported study in which this combination of techniques has been used to understand the interactions of an aphid and plant under different environmental conditions.     

Growth and translocation of C and N in wheat (Triticum aestivum) grown with a split root system

The root system of wheat seedlings ( Triticum aestivum L. SUN 9E) was pruned to two seminal roots. One of the roots was supplied with different levels of NO₃, the other was deprived of N. Root respiration and the increment of C and N in roots and shoots were measured to determine the C/N ratio of the phloem sap feeding the N-deprived roots. Thus it was possible to determine translocation of N from the shoots to the roots. It was calculated that the C/N ratio of phloem sap feeding roots of plants growing at optimal and suboptimal N supply was ca 54. A supra-optimal N supply reduced, whilst shading increased, the C/N ratio of phloem sap. At optimal N supply 11% of all N transported to the shoots was retranslocated to the roots. Both a supra-optimal and a limiting N supply increased translocation of N back to the roots to 18% of the N translocated to the shoot, whilst shading of the plants decreased the proportion cycled to 7%. At the optimal N supply, 40% more N was translocated to the roots from the shoot than was incorporated by them. At a lower supply of N, 80% more N was imported from the shoots than was incorporated by these roots. It is suggested that the distribution of N between roots and shoots predominantly occurs in the shoots. The specific mass transfer rate in seminal roots was determined. The highest value was found for roots grown with an optimal N supply: 1.1 mg carbohydrate s⁻¹ cm⁻² (sieve tube) which is well within the range observed for other plant organs. Roots supplied with NO₃ produced more and longer laterals than N-deprived roots. It is suggested that this is due to the effect of NO₃ on import of carbon and other components transported in the mass flow with carbon.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Changes in the rooting inhibitory effect of chestnut extracts during cold storage of the cuttings

Extracts of cold stored chestnut cuttings ( Castanea sativa Mill.) were examined for the inhibitory effect on the stimulation of rooting by 1AA which has been detected previously in extracts from freshly collected cuttings. The extracts were fractionated by paper chromatography and the different zones of the chromatograms were bioassayed together with 1AA by the bean rooting test. The bean rooting test showed that the inhibitory effect decreased with the length of cold storage period, so that after 5 months of storage, the inhibitory effect had disappeared, and a root promoting zone was found on the chromatograms. A comparative study of phenolics in this zone, before and after cold storage, revealed the formation of vanillyl and salicyl alcohols in the chilled material. Vanillyl and salicyl alcohols are rooting stimulators and increase the effect of 1AA on rooting in bean cuttings.     

Effect of nitrogen nutrition on amino acid composition of xylem sap and stem wood in Alnus glutinosa

Citrulline was the major amino acid in root pressure sap, stem sap and stem wood from Alnus glutinosa L. Gaertn. plants relying on fixed nitrogen or, partly or wholly, on mineral nitrogen for growth. Glutamine increased in prominence in plants assimilating mineral nitrogen but asparagine remained a relatively insignificant component. Differences in the relative amounts of the free amino compounds of stem sap from nitrogen-fixing and mineral nitrogen-fed plants were usually small compared to differences between plants fed different sources of mineral nitrogen. In contrast, relatively high values for the ratios of citrulline/total free amino nitrogen compounds and particularly of citrulline/amides in root pressure sap distinguished nitrogen-fixing plants from those receiving mineral nitrogen. Although the amino acid ratios of stem wood extracts showed closer similarity to those for root pressure sap than stem sap, the seasonal accumulation of citrulline, possibly as a storage amino acid, in stem wood from field-grown plants negated the possibility of utilising stem wood analyses as an indicator of the form of nitrogen assimilation. Comparative data on the levels of citrulline or other free amino acids in Alnus glutinosa are unlikely to be useful as an index of nitrogen fixation, under most experimental conditions.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Changes in peroxidase isoenzymes of Phaseolus mungo hypocotyl cuttings during rooting

A time course study of changes in the pattern of peroxidase isoenzymes shows that two new isoenzymes a and b appeared in hypocotyl cuttings in     

Phloem loading and unloading of sugars and amino acids

In terrestrial higher plants, phloem transport delivers most nutrients required for growth and storage processes. Some 90% of plant biomass, transported as sugars and amino nitrogen (N) compounds in a bulk flow of solution, is propelled though the phloem by osmotically generated hydrostatic pressure differences between source (net nutrient export) and sink (net nutrient import) ends of phloem paths. Source loading and sink unloading of sugars, amino N compounds and potassium largely account for phloem sap osmotic concentrations and hence pressure differences. A symplasmic component is characteristic of most loading and unloading pathways which, in some circumstances, may be interrupted by an apoplasmic step. Raffinose series sugars appear to be loaded symplasmically. However, sucrose, and probably certain amino acids, are loaded into minor veins from source leaf apoplasms by proton symporters localized to plasma membranes of their sieve element/companion cell (se/cc) complexes. Sucrose transporters, with complementary kinetic properties, are conceived to function as membrane transporter complexes that respond to alterations in source/sink balance. In contrast, symplasmic unloading is common for many sink types. Intervention of an apoplasmic step, distal from importing phloem, is reserved for special situations. Effluxers that release sucrose and amino acids to the surrounding apoplasm in phloem loading and unloading are yet to be cloned. The physiological behaviour of effluxers is consistent with facilitated membrane transport that can be energy coupled. Roles of sucrose and amino acid transporters in phloem unloading remain to be discovered along with mechanisms regulating symplasmic transport. The latter is hypothesized to exert significant control over phloem unloading and, in some circumstances, phloem loading.     

Effects of Acetylcholine, Cytochalasin B and Amiprophosmethyl on Phloem Transport in Radish （Raphanus sativas）

We investigated the role of the ＂sieve tube-companion cell complex＂ lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement （acetylcholine, （ACh） a neurotransmitter in animals and insects; cytochalasin B, （CB） a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, （APM） a specific inhibitor of many cellular responses that are mediated by microtubule systems）. Their effects on phloem transport were estimated by two experimental devices： （i） a comparison of changes in the amount of assimilates in terms of carbohydrates and ^14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and （ii） distribution patterns of ^14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations （5.0×10^-4 mol/L） but inhibited it in higher concentrations （2.0×10^-3 mol/L） from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Landscape patterns of free amino acids in arctic tundra soils

Concentrations of free amino acids were measured in soils from four major ecosystem types in arctic Alaska. Total free amino acid concentrations were several-fold higher than ammonium (the major form of inorganic nitrogen) in water extracts of soils. The dominant free amino acids in these soils were glycine, aspartic acid, glutamic acid, serine, and arginine. Concentrations of total amino acids ranged 5-fold across communities, being highest in tussock tundra and lowest in wet meadows. Incubation experiments indicate that the turnover of amino acids is rapid, which suggests high rates of gross nitrogen mineralization in these soils. The high concentrations and dynamic nature of soil free amino acids suggest that this nitrogen pool is a significant component of nitrogen cycling in these tundra ecosystems.     

High altitude acclimatization in fourArtemisia species: Changes in free amino acids and nitrogen contents in leaves

During high altitude acclimatization the highest number of amino acids were found in temperateArtemisia species (A. vestita) and the lowest one in tropical species (A. scoparia). The amount of free amino acids in temperateArtemisia species was higher when this was grown at 3600 m altitude. InA. scoparia, A. vulnaris andA. parviflora, the higher amount of individual amino acids was ascertained in plants grown at lower altitudes. InA. vestita, the nitrogen contents per unit dry matter was lower in plants grown at high altitude. In the other three species, the contents were significantly higher in the leaves of plants grown at a higher altitude. The nitrogen contents per unit leaf dry matter determined during active growth of plants were minimum inA. parviflora and maximum inA. vestita. With progressing plant senescence the concentration of nitrogen decreased in all species studied.