Measurement of xylem sap amino acid concentrations in conjunction with whole tree transpiration estimates spring N remobilization by cherry (Prunus avium L.) trees

Prunus avium trees were grown in sand culture for one vegetative season with contrasting N supplies, in order to precondition their N storage capacities. During the spring of the second year a constant amount of ¹⁵N was supplied to all the trees, and the recovery of unlabelled N in the new biomass production was used as a direct measure of N remobilization. Destructive harvests were taken during spring to determine the pattern of N remobilization and uptake. Measurements of both xylem sap amino acid profiles and whole tree transpiration rates were taken, to determine whether specific amino acids are translocated as a consequence of N remobilization and if remobilization can be quantified by calculating the flux of these amino acids in the xylem. Whereas remobilization started immediately after bud burst, N derived from uptake by root appeared in the leaves only 3 weeks later. The tree internal N status affected both the amount of N remobilization and its dynamics. The concentration of xylem sap amino acids peaked shortly after bud burst, concurrently with the period of fastest remobilization. Few amino acids and amides (Gln, Asn and Asp) were responsible for most of N translocated through the xylem; however, their relative concentration varied over spring, demonstrating that the transport of remobilized N occurred mainly with Gln whereas transport of N taken up from roots occurred mainly with Asn. Coupling measurements of amino acid N in the xylem sap with transpiration values was well correlated with the recovery of unlabelled N in the new biomass production. These results are discussed in relation to the possibility of measuring the spring remobilization of N in field‐grown trees by calculating the flux of N translocation in the xylem.     

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.     

Amino acid content in xylem sap of regrowing alfalfa (Medicago sativa L.): Relations with N uptake,N2 fixation and N remobilization

During vegetative regrowth of Medicago sativa L., soil N, symbiotically fixed N₂ and N reserves meet the nitrogen requirements for shoot regrowth. Experiments with nodulated or non-nodulated plants were carried out to investigate the changes in N flows originating from the different N sources and in xylem transport of amino acids during regrowth. Exogenous N uptake, N₂ fixation and endogenous N remobilization were estimated by ¹⁵N labelling and amino acids in xylem sap were analysed. Removal of shoots resulted in great declines of exogenous N flows derived either from N₂ or from NH₄NO₃ during the first week of regrowth, thereafter recovery increased linearly. Mineral N uptake as well as N₂ fixation occurred mainly between the 10th and 18th day after removal of shoots while exogenous N assimilation in intact plants remained at a steady level. Nitrogen remobilization rates in defoliated plants increased by at least three to five-fold, especially during the first 10 days following shoot removal. Compared to control plants, contents of amino acids in xylem sap, during the first 10 days of regrowth, were reduced by about 72% and 82% in NH₄NO₃ grown and in N₂ fixing plants, respectively. Asparagine was the main amino acid transported in xylem sap of both treated plants. Its relative contents during this period significantly decreased from 75% to 59% and from 67% to 36% respectively in non-nodulated plants and in nodulated ones. This decline was accompanied by compensatory increase in the relative contents of aspartate and glutamine.     

Dependence of amino acid composition upon nitrogen availability in birch (Betula pendula)

Small birch plants ( Betula pendula Roth .) were grown at different rates of exponentially increasing nitrogen supply. This resulted in plants with different relative growth rates and different internal nitrogen concentrations. Within a nitrogen treatment, both of these variables remained constant with time.
Free amino acids were measured in leaves and roots of the seedlings at two different harvests. At greater nitrogen supply, higher concentrations of total amino acid nitrogen were found in roots and leaves. The ratio of amino acid nitrogen to total nitrogen was low albeit greater at higher nitrogen supply. Higher concentrations of amino acid nitrogen were mainly due to high concentrations of citrulline, glutamine, γ-aminobuitric acid and arginine.
Greater leaf concentrations of amino acid nitrogen at higher nitrogen supply may be related lo increased concentrations in the xylem sap and/or may be indicative of small excesses of nitrogen with respect to current nitrogen usage in protein synthesis.     

Growth models of silver birch (Betula pendula Roth.) on two volcanic mountains in the French Massif Central

Silver birch woodlands of two volcanic mountains (altitude 850 m and 1450 m) were studied in order to establish a growth model of birch. Height, radial increment and crown width were measured on both sites taking into account two situations: isolated birch or birch within a stand. For the latter case two categories were sampled considering the competition status of the tree: birch suffering the most severe competition (suppressed tree) were distinguished from trees facing the lowest competition (dominant tree). Measures of competition were also made using plots where each tree was located by its coordinates and its height, trunk circumference and crown width recorded. Examination of radial growth curves showed similar patterns for the two sites for the same category of tree. Radial growth was always inferior for the site located at the highest altitude but tree lifespan was about two-fold longer. Radial increment data were used to calculate circumference as function of tree age. Models predicting circumference with time were then established for each category of tree (dominant, suppressed or isolated) using Chapman–Richards'model. Height and crown width models were produced using circumference as the predictor. Competition indices based on vertical or horizontal angles weighted by the distance were calculated for birches in stands. Indices using vertical angles appeared to be more resistant and robust to characterize local competition. Competition index was then associated with the parameters of Chapman-Richards' growth circumference model for the two sites and models predicting the circumference from the age of the tree and its competition index are proposed.     

Evidence for discontinuous water columns in the xylem conduit of tall birch trees

The continuity of the xylem water columns was studied on 17- to 23-m tall birch trees (trunk diameter about 23 cm; first branching above 10 m) all year round. Fifty-one trees were felled, and 5-cm thick slices or 2-m long boles were taken at regular, relatively short intervals over the entire height of the trees. The filling status of the vessels was determined by (i) xylem sap extraction from trunk and branch pieces (using the gas bubble-based jet-discharge method and centrifugation) and from trunk boles (using gravity discharge); (ii) ¹H nuclear magnetic resonance imaging of slice pieces; (iii) infusion experiments (dye, ⁸⁶Rb⁺, D₂O) on intact trees and cut branches; and (iv) xylem pressure measurements. This broad array of techniques disclosed no evidence for continuous water-filled columns, as postulated by the Cohesion–Tension theory, for root to apex directed mass transport. Except in early spring (during the xylem refilling phase) and after extremely heavy rainfall during the vegetation period, cohesive/mobile water was found predominantly at intermediate heights of the trunks but not at the base or towards the top of the tree. Similar results were obtained for branches. Furthermore, upper branches generally contained more cohesive/mobile water than lower branches. The results suggest that water lifting occurs by short-distance (capillary, osmotic and/or transpiration-bound) tension gradients as well as by mobilisation of water in the parenchymatic tissues and the heartwood, and by moisture uptake through lenticels.     

Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. II. Stomatal sensitivity to abscisic acid imported from the xylem sap

Sunflower plants [Helianthus annuus L.) were subjected to soil drought. Leaf conductance declined with soil water content even when the shoot was kept turgid throughout the drying period. The concentration of abscisic acid in the xylem sap increased with decreasing soil water content. No general relation could be established between abscisic acid concentration in the xylem sap and leaf conductance due to marked differences in the sensitivity of leaf conductance of individual plants to abscisic acid from the xylem sap. The combination of these results with data from Gollan, Schurr & Schulze (1992, see pp. 551–559, this issue) reveals close connection of the effectiveness of abscisic acid as a root to shoot signal to the nutritional status of the plant.     

Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. I. The concentration of cations, anions, amino acids in, and pH of, the xylem sap

Sunflower plants (Helianthus annuus L.) were subjected to soil drying with their shoots either kept fully turgid using a Passioura-type pressure chamber or allowed to decrease in water potential. Whether the shoots were kept turgid or not, leaf conductance decreased below a certain soil water content. During the soil drying, xylem sap samples were taken from individual intact and transpiring plants. Xylem sap concentrations of nitrate and phosphate decreased with soil water content, whereas the concentrations of the other anions (SO₄² and Cl^?) remained unaltered. Calcium concentrations also decreased. Potassium, magnesium, manganese and sodium concentrations stayed constant during soil drying. In contrast, the pH, the buffering capacity at a pH below 5 and the cation/anion ratio increased after soil water content was lowered below a certain threshold. Amino acid concentration of the xylem sap increased with decreasing soil water content. The effect of changes in ion concentrations in the xylem sap on leaf conductance is discussed.     

Changes in the amide and amino acid composition of xylem exudate from perennial ryegrass (Lolium perenne L.) during regrowth after defoliation

The paper presents the results of amino acid analyses in xylem sap during leaf regrowth of ryegrass plants defoliated firstly at the 8th and secondly at the 12th week of culture. The free amino acid composition of leaves, stubble and roots was also determined and some of the results are reported. Prior to defoliation, xylem sap contained a high proportion of amides, particularly glutamine. During regrowth after defoliation, the proportion of asparagine in the xylem sap increased until the third day when the highest ratios of asparagine/glutamine appeared. The results are compared with relative amounts of free amino acids in the different plant parts and discussed in relation to source-sink nitrogen transfer.     

A novel approach to the in situ measurement of oxygen concentrations in the sapwood of woody plants

A novel technique for the physico‐chemical analysis of xylem sap by underwater access to the sapwood of trees is described. In situ measurements of dissolved oxygen in the sapwood are performed by combining this technique with a novel optical method for oxygen detection. In early spring, the oxygen concentration of the sapwood of Betula pendula was in the range of 80–230 µmol O₂ L^?1, corresponding to an oxygen deficit of 40–75% of air saturation. Oxygen concentration maxima and minima occurred early in the morning and in the afternoon, respectively, whereas xylem sap temperatures showed the reverse pattern. In the sapwood, hypoxia increased from the beginning of bud break until frondescence, when a deficit of 86% of air saturation marked the upper limit of oxygen depletion. There seemed to be no relationship between daily variations of oxygen concentration and xylem sap pressure. In summer, sap flow was a major determinant for the diurnal variation of dissolved oxygen concentration. Oxygen supply to the sapwood was determined by both radial influx into the trunk through intercellular gas spaces and transport of dissolved oxygen via xylem sap flow. Radial influx seemed to be favoured during night‐time, when the trunk was warmer than ambient air. During daytime, the hypoxia of the sapwood rose and increased sharply in the evening, when sap flow velocity approximated zero. High temperature in the sapwood enhanced the respiratory oxygen consumption of the wood parenchyma while the supply of dissolved oxygen via the transpiration stream became ineffective.     

Influence of aluminum and phosphorus on growth and xylem sap composition in Melastoma malabathricum L.

Melastoma (Melastoma malabathricum L.) is an aluminum-accumulating woody plant that accumulates more than 10 000 mg kg^–1 of aluminum (Al) in mature leaves. The influence of Al and phosphorus (P) applications on plant growth and xylem sap was examined in the present study in order to elucidate the interaction between Al-induced growth enhancement and P nutrition, and to determine the form of Al for translocation from roots to shoots. Although the Al application significantly increased the growth of Melastomaseedlings with the high P pre-treatment, and P concentrations in the leaves and Pi concentrations in the xylem sap regardless of the P pre-treatment, we could not come to the conclusion that a primary cause of the Al-induced growth enhancement in Melastoma is the stimulation of P uptake. The degree of Al-induced growth enhancement corresponded not with the P concentrations but with the Al concentrations in the plant tissue, suggesting that the Al-induced growth enhancement in Melastoma is primarily caused by Al itself in the plant tissue rather than by the stimulation of P uptake. Through the analysis of organic acids and Al in the xylem sap and plant tissue, the form of Al for translocation from roots to shoots was shown to be an Al-citrate complex that was transformed into Al-oxalate complex for Al storage in the leaves. In addition, the xylem sap of Melastoma seedlings grown in the absence of Al contained higher concentrations of malate. In the presence of Al, however, higher concentrations of citrate were found, indicating that Melastoma changes its organic acid metabolism in the presence or absence of Al; more specifically, it increases the synthesis of citrate.     

N content of phloem and xylem exudates during the transition to flowering in Sinapis alba and Arabidopsis thaliana

The involvement of nitrogenous substances in the transition to flowering was investigated in Sinapis alba and Arabidopsis thaliana (Columbia). Both species grown in short days (SD) are induced to flower by one long day (LD). In S. alba, the phloem sap (leaf and apical exudates) and the xylem sap (root exudate) were analysed in LD versus SD. In A. thaliana, only the leaf exudate could be analysed but an alternative system for inducing flowering without day‐length extension was used: the displaced SD (DSD). Significant results are: (i) in both species, the leaf exudate was enriched in Gln during the inductive LD, at a time compatible with export of the floral stimulus; (ii) in S. alba, the root export of amino acids decreased in LD, whereas the nitrate remained unchanged – thus the extra‐Gln found in the leaf exudate should originate from the leaves; (iii) extra‐Gln was also found very early in the apical exudate of S. alba in LD, together with more Glu; (iv) in A. thaliana induced by one DSD, the leaf export of Asn increased sharply, instead of Gln in LD. This agrees with Asn prevalence in C‐limited plants. The putative role of amino acids in the transition to flowering is discussed.     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr.

Comparative analyses of aspects of the carbon (C) physiology and the expression of C transporter genes in birch (Betula pendula Roth.) colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. were performed using mycorrhizal (M) and non‐mycorrhizal (NM) plants of similar foliar nutrient status. After six months of growth, the biomass of M plants was significantly lower than that of NM plants. Diurnal C budgets of both sets of plants revealed that M plants exhibited higher rates of photosynthesis and root respiration expressed per unit dry weight. However, the diurnal net C gain of M and NM plants remained similar. Ectomycorrhizal roots contained higher soluble carbohydrate pools and increased activity of cell wall invertase, suggesting that additional C was allocated to these roots and their ectomycorrhizal fungi consistent with an increased sink demand for C due to the presence of the mycobiont. In M roots, the expression of two hexose and one sucrose transporter genes of birch were reduced to less than one‐third of the expression level observed in NM roots. Analysis using a probe against the birch ribosomal internal transcribed spacer region revealed that M roots contained 22% less plant RNA than NM roots. As the expression of birch hexose and sucrose transporter genes was reduced to a much greater extent, this suggests that these specific genes were down‐regulated in response to alterations in C metabolism within M roots.