Spring Mobilization of Storage Nitrogen in Isolated Shoot Section of Apple

The process of mobilization of nitrogenous compounds in trees during spring development was studied in short isolated shoot sections (usually bearing one bud each) of Golden Delicious apple trees. During leafing-out of the bud, changes in the amounts of total, protein and soluble nitrogen and of soluble amino acids and amides in bark and wood were followed. The nitrogen required by the growing parts came mainly from protein breakdown in the tissues below the bud; in the tissues above the bud, total nitrogen decreased little, whereas the drop in protein nitrogen was considerable. In de-budded sections and in internode sections where total nitrogen remained almost unchanged, protein hydrolysis occurred as well. It is concluded that the protein breakdown is not strongly dependent on the demand of the bud for nitrogen. Inversion of the sections did not result in any change in the pattern of nitrogen mobilization: a marked drop occurred in the nitrogen content of the physiologically basal part of the section and only a slight decrease in the apical part. The translocation of stored nitrogenous compounds to the growing parts seems to occur in the phloem, at least over short distances. Asparagine and arginine were found to be the major components of the soluble amino-nitrogen fraction throughout. The relative importance of asparagine was reduced in tissue where a substantial loss of nitrogen occurred during leafing-out of the bud. This is explained in terms of a preferential export of asparagine to the bud.     

Spring Mobilization of Protein Nitrogen in Apple Bark

Mobilization of protein nitrogen in the spring was studied in bark of stems of unringed and double stem-ringed apple rootstocks M.7 given different nitrogen treatments. A ready protein hydrolysis occurred; the proteins contributed the greater part of the storage nitrogen exported to the growing parts. Protein hydrolysis was little affected by the supply of newly absorbed nitrogen. Movement of nitrogen out of the bark between the rings could not be demonstrated. Protein breakdown in the isolated bark sections was slightly reduced. Arginine was the predominant amino acid in the proteins of the trees with a high level of storage nitrogen but was not conspicuous in the low-nitrogen trees. The protein composition changed little during hydrolysis. Only the share of arginine in the high-nitrogen trees dropped appreciably. It is suggested that the high-nitrogen trees possess a special storage protein characterized by a high arginine content. Analysis of the nitrogen fraction of isolated bark sections showed that the composition of the soluble nitrogen was characterized by a high level of asparagine and especially of arginine, and was quite different from the composition of the proteins. The data suggest that the asparagine in particular originated largely from transformation of the various amino acids set free during protein hydrolysis.     

Uptake and Distribution of Nitrogen in Young Apple Trees after Application of Nitrate or Ammonium, with Special Reference to Asparagine and Arginine

Apple rootstocks M.7 were given either nitrate or ammonium at the end of September. Until the following June, total and protein nitrogen and the composition of the soluble nitrogen fraction were followed in the roots, stem-bark, and new growth. Nitrogen from both sources was readily absorbed in roughly the same amounts. Absorption occurred in the autumn and especially in the following spring. Incorporation of the absorbed nitrogen took place exclusively in the young roots. Arginine and, to a lower degree, asparagine were by far the most abundant of the soluble amino compounds and reached levels far above those in the unfertilized trees. The asparagine/arginine ratio was rather high during the first few weeks after fertilization but then dropped steadily to a low winter level. During May and June there was a gradual decrease to very low levels, especially in the new growth. The data suggest that the absorbed nitrogen is incorporated into asparagine and that arginine is formed from asparagine if the supply of nitrogen exceeds the immediate needs. Throughout the experimental period, ammonium fertilization led to higher values of the asparagine/arginine ratio than did nitrate nutrition. This is explained in terms of an enhanced production of asparagine and a retarded conversion into arginine. It is argued that asparagine is the main translocation compound for nitrogen. The possibility is discussed that, in addition, arginine moves upwards by a process of exchange along the negatively charged walls of the xylem vessels.     

Effect of root temperature on the amino-nitrogen composition of leaves in successive segments of apple shoots

Young apple trees ( Malus pumila Mill. cv. Cox's Orange Pippin) given nitrogen either at or 40 days after bud-break were kept at a root temperature of 6, 18 or 30°C under otherwise constant conditions. Twelve weeks after the start of the experiment leaves from successive shoot segments and roots were collected and in most cases analysed to assess total nitrogen, protein nitrogen, and the main amides and amino acids. The percentage composition of the amino-nitrogen fraction of the roots was not or was hardly at all affected by the treatments; asparagine predominated, followed by arginine. In contrast, in the leaves the share of arginine dropped from about 90% at 6°C to about 30% at 30°C in favour of especially asparagine. This pattern was mainly attributable to the situation in the basal sections of the shoot. In the middle and top segments the temperature effects were small. In general, a high level of amino nitrogen corresponded to a high contribution of arginine. Soluble nitrogen was higher after the late than after the early application of nitrogen. Shoot growth was reduced at 6°C root temperature, but little difference was seen between 18 and 30°C. It was concluded that with respect to nitrogen metabolism roots and shoots function more or less independently of each other. The hypothesis that the roots affect leaf nitrogen metabolism via the supply of growth substances produced in the roots, presumably cytokinins, is discussed.     

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.     

Changes in Total Nitrogen and Free Amino Acids in Stem Cuttings of Mulberry (Morus alba L.)

Changes in total nitrogen and free amino acid contents in stemcuttings of Morus alba have been studied. The fresh and dryweights and total nitrogen amounts of the parent stems of cuttingsdecreased initially after cutting. Their increase follows theformation of main roots in cuttings, suggesting that, like carbohydrates,sugars and starch, stored nitrogenous substances are used forsprouting and rooting of cuttings. Amino acids found in stems,roots and shoots are those common in other higher plants withthe exception of pipecolic acid and 5-hydroxypipecolic acid.Significant changes in the levels of asparagine, proline, arginine,-aminobutyric acid and alanine in roots, bark and wood of parentstems were observed during cutting growth, whereas those ofother amino acids remained comparatively constant; the mostpredominant amino acid in the starting materials was proline.while that in the cuttings during growth was asparagine. Theresults suggest that, among free amino acids, asparagine, prolineand arginine play the major part in storage of nitrogen in mulberry.The importance of glut-amine and asparagine in nitrogen metabolismin mulberry has been discussed.     

Nitrogen and Cation Nutrition of Three Ecologically Different Plant Species

Apple rootstocks M.7 were given a nitrogen application either in the spring or in the preceding autumn. At the time of the spring application some rootstocks were ringed. During the 50-day experimental period from bud-break, shoot growth and the amount of nitrogen incorporated into the new shoots were slightly reduced in the spring-treated trees and strongly reduced in the ringed trees of both treatments. Roots of unringed autumn-fertilized trees showed higher levels of total and amino nitrogen than those of similar trees in the spring treatment; to a lesser degree, the reverse held for xylem sap from the stem. Ringing increased the amino-nitrogen level in the roots, which suggests a reduced translocation rate. The nitrogen treatments led to marked differences in the percentage composition of the amino-nitrogen fraction of roots and xylem sap. The distribution of amino acids and amides in the roots and that in xylem sap of the same trees was divergent, but arginine and asparagine often were the most important constituents. Aspartic acid was rather abundant in xylem sap. Ringing did not affect the composition of the amino-nitrogen fraction in the roots quantitatively but increased the proportion of arginine in the sap. The possible relationship between the composition of xylem sap and soluble nitrogen in the roots is discussed. It is argued that especially in spring-fertilized trees appreciable amounts of nitrogen must be translocated via the phloem in addition to the transport in the xylem.     

Seasonal variation of nitrogenous compounds in the xylem sap of Citrus

Samples of tracheal sap of Citrus sinensis (L.) Osbeck cv. Washington Navel were taken from field trees throughout the year and the nitrogen composition of the sap was determined. The nitrogenous fraction of the sap was composed mainly of free amino acids (92–97% of total nitrogen) and nitrates throughout the year. Proline was the most abundant amino acid during almost the entire cycle, and its concentration was especially high during the autumn and winter period. Nevertheless, a significant part (40–60%) of the total organic nitrogen was transported as arginine. Total nitrogen as well as amino acids and nitrates were maximal at spring flush. At spring flush and summer flush there was also a diversification of α-amino nitrogen among different amino acids. During the spring flush, nitrates, asparagine and γ-aminobutyric acid in the xylem sap seemed to have a radicular origin, whereas glutamic acid and arginine were released from the surrounding parenchyma. The results suggest a metabolic transformation in the wood parenchyma of nitrogenous compounds coming from the roots (including reduction of nitrates) and a turnover of different nitrogen metabolites between the xylem and surrounding cells.     

Environmental factors affecting basic nitrogen metabolism and seasonal levels of various nitrogen fractions in tissues of bilberry, Vaccinium myrtillus

Bilberry tissues accumulated nitrogen for the winter in the form of reduced low-molecular weight amino compounds. The storage organ was principally the underground stem and the oldest parts of the aerial shoot. Most of the nitrogen was stored in arginine and ammonium compounds, and less in glutamine and other amino acids. Proteins did not accumulate during the winter. The soluble nitrogenous compounds were discharged from storage in May, when nitrogen was translocated from the lower parts of the stem to the growing leaves and buds. Amino acid compositions and concentrations in winter were almost identical under the snow and in snowless areas, only the concentration of glutamine being lower and that of glutathione higher in the snowless area. The level of total protein, particularly in the leaves and buds was much higher in a nitrogen-polluted industrial area than in unpolluted urban forests. The same difference was observed in total amino compounds, but among individual substances it only appeared in ammonium compounds. Certain species differences in the amino acid pool were recorded between V. myrtillus and V. vitis-idaea.     

Changes in nitrogen levels and free amino acids in rooting cuttings of mulberry (Moms alba)

Total and protein nitrogen in bark and wood of parent stems of mulberry ( Morns alba L. cv. Ichinose) decreased readily and to the same extent during leafing-out of the buds, but the decrease in wood was less marked than in bark. Simultaneously, soluble nitrogen in both bark and wood also declined but the depletion was less marked than that of total and protein nitrogen. During the same period total nitrogen in the new shoots and adventitious roots increased drastically; however, the increase in total nitrogen in the growing parts during rooting was almost the same as the decrease in total nitrogen in the parent stems. Proline, the prevalent amino acid in wood and bark of the parent stems, decreased drastically during rooting, whereas during the same period asparagine in the developing buds, callus and adventitious roots increased markedly and became the predominant amino acid. The amount of arginine was relatively high in bark of the parent stems but Low in wood and the buds. The level of arginine in bark decreased considerably during the experiments (as did that of proline). The results suggest that the nitrogen required by the growing parts (sinks) in the rooting cuttings comes mainly from protein breakdown in bark of the parent stems (source), although stored protein in wood (source) and soluble nitrogen in bark and wood (sources) also play a part in storage of nitrogen. Asparagine is suggested to be the main nitrogen transport compound in the new growth of the tree and the initiating roots of cuttings.     

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.     

The influence of the cooling of donor cultures on the in vitro adventitious regeneration and carbohydrate metabolism of four dwarfing apple rootstocks

We examined the effects of cooling applied for 4 to 20 weeks on donor cultures of four dwarfing apple rootstocks (P16, P22, P59 and M26). Our aim includes increasing their competence for in vitro adventitious shoot regeneration from the leaves. Donor cultures were maintained on a shoot multiplication medium at 4°C in the dark for 4 months, followed by subculture on a fresh medium for 4 weeks. The cooling of the cultures caused an increase in the adventitious shoot number and a decrease in the starch content and an increase in the soluble sugar content (monosaccharides, raffinose and stachyose). The accumulation of stachyose in response to cold is a new observation, and it suggests that raffinose and stachyose play important role in the acclimation of dwarf apple rootstocks to low temperatures.     

Major nitrogen compounds transported in xylem vessels from roots to top in Citrus trees

Four-year-old citrus trees ( Citrus unshiu Marcovitch) were fed via the roots with (¹⁵NH₄)2sO₄ or K¹⁵NO₃ as a nitrogen source. Nitrogenous compounds and their isotopic abundances in fine roots and xylem sap from trunks were assayed in order to obtain information on the species of nitrogen released by the root system into the ascending xyiem stream.
Arginine, asparagine, nitrate and proline in xylem sap accounted for 48, 21, 13 and 10%, respectively, of the total nitrogenous constituents tested in the sap. However, in the trees fed with labelled ammonium the main nitrogenous compound labelled with ¹⁵N in the xylem sap was asparagine and glutamine, which accounted for 79% and 18%, respectively, of total labelled nitrogen. In the xylem sap of trees fed with labelled nitrate, nitrate accounted for 94% of total labelled nitrogen. Nitrate and asparagine followed by glutamine showed the highest ratios of isotopic abundance in xylem sap as compared to fine roots. Proline and arginine had much lower ratios. These results indicate that nitrate, asparagine and glutamine are the main nitrogenous compounds released by the roots to the xylem stream, whereas arginine and proline are released into the xylern vessels by the trunk tissues. Furthermore, nitrate and asparagine are probably in steady movement upward in the trunk xylem, whereas glutamine is more easily taken up by the trunk tissues than nitrate and asparagine.     

Organic nitrogen reserves and their mobilization during sprouting of purple nutsedge (Cyperus rotundus L.) tubers

Purple nutsedge is a perennial weed propagating vegetativelyby an extended network of rhizomes and tubers. Large quantitiesof starch are stored in the tubers, but the properties of nitrogenreserves and their importance for early plant growth have receivedlittle attention in the past. Organic nitrogen compounds werestudied in mature tubers by protein determination, SDS-PAGEand amino acid quantification and separation using reversed-phaseHPLC. Changes in these compounds were followed in sproutingtuber pieces fed with a complete nutrient solution (containingnitrate, control) or with a nutrient solution without nitrogensource. As judged by gel electrophoresis or direct protein quantification,some net protein degradation occurred after 2–4 weeksof sprouting in the presence or absence of exogenous N. Aminoacids decreased much faster, especially during the first 2 weeks.The major amino acids were arginine and asparagine, which togetheraccounted for 70% of total amino acids at day 0, and which hadalmost disappeared after 4 weeks of sprouting. Sprout growthdepended strongly on the availability of exogenous nitrogenduring the second week. The results indicate that amino acidsare the main N storage form of purple nutsedge tubers and, essentially,that no storage proteins are present. Future investigationson the N metabolism of sprouting nutsedge should focus on arginineand asparagine metabolism and eventually on nitrogen assimilation,which becomes important in an early phase of plant growth. Key words: Cyperus rotundus L., arginine, asparagine, nitrogen assimilation, storage proteins     

Amino acid pattern and glutamate metabolism during dehydration stress in the 'resurrection' plant Sporobolus stapfianus: a comparison between desiccation-sensitive and desiccation-tolerant leaves

The present study analyses changes in nitrogen compounds, amino acid composition, and glutamate metabolism in the resurrection plant Sporobolus stapfianus during dehydration stress. Results showed that older leaves (OL) were desiccation-sensitive whereas younger leaves (YL) were desiccation-tolerant. OL lost their soluble protein more rapidly, and to a larger extent than YL. Enzymes of primary nitrogen assimilation were affected by desiccation and the decrease in the glutamine synthetase (GS, EC 6.3.1.2) and ferredoxin-dependent GOGAT (Fd-GOGAT, EC 1.4.7.1) activities was higher in OL than in YL, thus suggesting higher sensibility to dehydration. Moreover, YL showed higher total GS enzyme activity at the end of the dehydration stress and was shown to maintain high chloroplastic GS protein content during the entire stress period. Free amino acid content increased in both YL and OL between 88% and 6% relative water content. Interestingly, OL and YL did not accumulate the same amino acids. OL accumulated large amounts of proline and gamma-aminobutyrate whereas YL preferentially accumulated asparagine and arginine. It is concluded (i) that modifications in the nitrogen and amino acid metabolism during dehydration stress were different depending on leaf development and (ii) that proline and gamma-aminobutyrate accumulation in S. stapfianus leaves were not essential for the acquisition of desiccation tolerance. On the contrary, the accumulation of large amounts of asparagine and arginine in the YL during dehydration could be important and serve as essential nitrogen and carbon reservoirs useful during rehydration. In this context, the role of GS for asparagine accumulation in YL is discussed.     

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.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

Extraction of tracheal sap from Citrus and analysis of its nitrogenous compounds

Tracheal sap was extracted from sections of stems (0.5 to 1.5 cm in diameter and 7.5 to 15.0 cm in length) of orange trees (Citrus sinensis (L.) Osbeck cv. Washington Navel) by using a combination of the vacuum and liquid displacement methods. The volume of sap obtained and its concentration of nitrogenous compounds were dependent on the volume of displacing liquid used for the extraction. Four ml of water-saturated 1-butanol extracted essentially all of the xylem fluid present in the stem sections without apparent production of artifacts. The time of sampling affected the nitrogen concentration of the tracheal sap, but not the content of xylem nitrogen per volume of stem material. The orientation of the stems in the tree and the diameter of the stems had an effect on their contents of xylem nitrogen, with southeastern orientation and thinner stems showing higher concentrations. We could not detect the presence of ammonium, nitrites or proteins in the tracheal sap of orange trees. Most of the nitrogen was present as amino acids and about 2% of the total in the form of nitrates. The qualitative composition of amino acids, as determined by TLC, was the same both in winter and spring tracheal sap. The main components of the sap were proline and arginine in winter, and these amino acids together with asparagine and aspartic acid in spring.     

GC-MS-SIM analysis of abscisic acid and indole-3-acetic acid in shoot bark of apple rootstocks

Concentrations of abscisic acid and indole-3-acetic acid were measured by GC-MS-SIM in the shoot bark of clonal apple rootstocks (M.27, M.9, MM.106 and MM.111) when the rootstocks were growing actively in the UK. These rootstocks are known to exhibit a wide range of control of tree size when grafted to a common scion. Shoot bark of the dwarfing rootstocks (M.27 and M.9) contained higher concentrations than the more vigorous rootstocks (MM.106 and MM.111) of ABA. Concentrations of ABA increased from May to July, followed by a decline in August. Only the month of sampling showed any significant influence on the concentration of IAA in shoot bark; however, there was a general increase, although not significant statistically, in IAA concentration with the increasing invigoration-capacity of the rootstock. At each sampling date the dwarfing rootstocks showed greater ratios of ABA:IAA than the invigorating rootstocks and generally the ratio for each rootstock increased from May to July, except for M.27 which showed the smallest ratio in June and the largest ratio in August. The results are discussed in relation to the generally accepted control exerted by the rootstocks on tree size and the possible influence of ABA on polar auxin transport.     

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.