Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.     

Effects of timing and supply of nitrogen on nitrogen remobilization from vegetative organs and redistribution to developing seeds of sunflower

A glasshouse study was made of the distribution of ¹⁵N among vegetative organs of sunflower and its later remobilization and redistribution to seeds, as influenced by the developmental stage at which ¹⁵N was provided, and by the N status of the plants. Plants of Hysun 30 sunflower were grown in sand culture and provided with K¹⁵NO₃ for a 3-day period at: (a) 3 days before the end of floret initiation; (b) 3 days before anthesis; (c) the start of anthesis; (d) full anthesis; and (e) 8 days after full anthesis. The plants were grown on a range of N supply rates, from severely deficient to more than adequate for maximum growth. Nitrogen-15 was distributed to all parts of the plant at the end of the ¹⁵N uptake periods. With the exception of the most N-stressed plants, subsequent remobilization of ¹⁵N from roots, stems and leaves occurred irrespective of the time the ¹⁵N was taken up. However, the percentage redistribution to seeds of ¹⁵N taken up at the end of floret initiation was less than for ¹⁵N taken up at anthesis. Remobilization of ¹⁵N from leaves and roots was higher (70%) for ¹⁵N taken up during and after anthesis than for ¹⁵N taken up at the end of floret initiation (45%), except for plants grown on the lowest N supply. By contrast, remobilization of ¹⁵N from the stem was lower for ¹⁵N taken up after full anthesis (40%) than before or during anthesis (>70%). The proportion of ¹⁵N remobilized from the top third of the stem was less than that from the bottom third, and decreased with increasing plant N status. Nitrogen-15 taken up over the 3-day supply periods during anthesis contributed from 2 to 11% of the total seed N at maturity; the contribution to seeds was greatest for plants grown on the highest N supply. Nitrogen taken up just before and during anthesis contributed most of the N accumulated in mature seeds of plants grown on an adequate N supply, but N taken up between the end of floret initiation and just before anthesis, or after full anthesis seemed to make an equally important contribution to mature seeds as N taken up during anthesis for plants grown on a very low N supply. It was concluded that the development of florets and seeds of sunflower is supported by N taken up by the plant between the end of floret initiation and anthesis, and by N redistributed from vegetative organs. Unless soil N is so low as to impair early growth, split applications of N fertilizer would be best made just before the end of floret initiation (‘star stage’) and just before anthesis.     

Estimating belowground nitrogen inputs of pea and canola and their contribution to soil inorganic N pools using 15N labeling

Background and aims

Crop species grown in a diversified crop rotation can influence soil N dynamics to varying degrees due to differences in the quantity and quality of the residues returned to the soil. The aim of this study was to quantify the contribution of N rhizodeposition by canola (Brassica napus L.) and pea (Pisum sativum L.) to the crop residue N balance and soil inorganic N pool.

Methods

Canola and pea were grown in a soil-sand mixture and were subject to cotton-wick ¹⁵N labeling in a greenhouse experiment. Nitrogen-15 recovered in the soil and roots were used to estimate N rhizodeposition.

Results

Belowground N, including root N and N rhizodeposits, comprised 70 % and 61 % of total crop residue N for canola and pea, respectively. Canola released the greatest amount of total root-derived N to the soil, which was related to greater root biomass production by canola. However, root-derived N in the soil inorganic N pool was greater under pea (13 %) than canola (4 %).

Conclusions

Our results show a significant belowground N contribution to total crop residue from pea and canola. Further investigation is required to determine whether input of the more labile N rhizodeposits of pea improves soil N supply to succeeding crops or increases the potential for N loss from the soil system relative to canola.     

Effect of N source during soybean pod filling on nitrogen and sulfur assimilation and remobilization

During pod filling, a grain legume remobilizes vegetative nitrogen and sulfur to its developing fruit. This study was conducted to determine whether different nitrogen sources affected N and S assimilation and remobilization during pod filling. Well-nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea assimilated 0%, 37%, or 114% more N, respectively, and 25%, 46%, or 56% more S, respectively, than did the average non-nodulated control plant fed 5.0 mM KNO₃. Thus, N source during pod filling greatly affected both N and S assimilation. Depending upon N source, plant N concentration during pod filling decreased from 2.96% to between 1.36% and 1.82%. Non-nodulated control plants fed 5.0 mM KNO₃ had the highest residual N at harvest. During the same treatments, plant S concentration decreased from 0.246% to a relatively uniform 0.215%. Thus, during pod filling, vegetative N was seemingly remobilized more efficiently (38–54%) than was S (13%). N source also affected seed yield and seed quality. Non-nodulated control plants fed 5.0 mM KNO₃ produced the lowest yield (21.1 g seeds plant^-1), whereas well nodulated plants fed 1.0 mM KNO₃, 0.5 mM urea, or 2.5 mM urea produced yields of 26.2 g, 31.8 g, and 36.7 g seeds plant^-1, respectively. Non-nodulated plants fed 2.5 mM urea yielded 28.6 g of seeds plant^-1. Seed N concentrations of non-nodulated plants and nodulated plants fed 2.5 mM urea were high, 6.30% and 6.11% N, respectively, whereas their seed S concentrations were low, 0.348% and 0.330% S, respectively. N sources that produced both a relatively high seed yield and seed N concentration (i.e., a relatively high total seed N plant^-1) produced a proportionately smaller increase in total seed sulfur. Consequently, seed quality, as judged solely by seed S concentration, was lowered.     

Effect of nitrogen on uptake, remobilization and partitioning of zinc and iron throughout the development of durum wheat

Deficiencies of zinc (Zn) and iron (Fe) are global nutritional problems and caused most often by their limited dietary intake. Increasing Zn and Fe concentrations of staple food crops such as wheat is therefore an important global challenge. This study investigated the effects of varied nitrogen (N) and Zn supply on the total uptake, remobilization and partitioning of Zn, Fe and N in durum wheat throughout its ontogenesis. Plants were grown under greenhouse conditions with high or low supply of N and Zn, and harvested at 8 different developmental stages for analysis of Zn, Fe and N in leaves, stems, husks and grains. The results obtained showed that the Zn and Fe uptake per plant was enhanced up to 4-fold by high N supply while the increases in plant growth by high N supply were much less. When both the Zn and N supplies were high, approximately 50% of grain Zn and 80% of grain Fe were provided by post-anthesis shoot uptake, indicating that the contribution of remobilization to grain accumulation was higher for Zn than for Fe. At the high N and Zn application, about 60% of Zn, but only 40% of Fe initially stored in vegetative parts were retranslocated to grains, and nearly 80% of total shoot Zn and 60% of total shoot Fe were harvested with grains. All these values were significantly lower at the low N treatment. Results indicate that N nutrition is a critical factor in both the acquisition and grain allocation of Zn and Fe in wheat.     

Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees

The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed.     

Glycine metabolism in intact leaves by in vivo 13C and 15N labeling

Solid-state (13)C NMR measurements of intact soybean leaves labeled by (13)CO(2) (at subambient concentrations) show that excess glycine from the photorespiratory C(2) cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as (13)C-labeled glycyl residues in proteins. This (13)C incorporation in leaf protein, which is uniformly (15)N labeled by (15)NH(4)(15)NO(3), occurs as soon as 2 min after the start of (13)CO(2) labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.     

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.     

Decorrelating source and sink determinism of nitrogen remobilization during grain filling in wheat

Background and Aims

Nitrogen (N) remobilization is the major source of N for grain filling in wheat, the other being N uptake after anthesis (N_up); however, variations in remobilization efficiency are not fully understood. It is hard to tell whether the source or the sink effects predominate, because N in the culm at anthesis (N_ant) correlates strongly with both N remobilization (N_rem) and grain number (G_n), respectively the main source and the main sink.

Methods

A pot experiment was thus designed to assess the relative contributions of the source and sink to N_rem regulation. Using two cultivars of winter wheat (Triticum aestivum, ‘Apache’ and ‘Autan’), three pre-anthesis and two post-anthesis N fertilization levels were applied in order to vary the N sources, while ear trimming at anthesis reduced sink size.

Key Results

Unlike results observed at a scale of m², the equation binding N_ant to N_rem exhibited a negative intercept, challenging the concept of nitrogen remobilization efficiency. Before ear trimming, G_n fitted well to N_ant, with a slope dependent on genotype. To obtain a sink variable that was less correlated with N_ant, the difference δG_n was calculated between actual grain number and that which could be predicted from culm N before trimming. A multiple regression then predicted N_rem (r² = 0·95) from N_ant, N_up and δG_n, with fitting unbiased by fertilization treatment, trimming or genotype.

Conclusions

In untrimmed culms, δG_n had a negligible effect, so that N_rem could be fitted to N_ant and N_up only: grain N filling appeared to be determined by sources only (N_ant and N_up), not by sink, and the reduction of N_rem by N_up was quantified. In these ‘normal’ cases, the regulation of N_rem should thus be located within the N sources themselves. In contrast, ear-trimming needs to be considered with caution as it introduced a sink limitation on N_rem; moreover one with an important genotype effect.Key words: Triticum aestivum, winter wheat, source/sink, grain filling, nitrogen uptake, grain number, nitrogen harvest index, nitrogen remobilization efficiency, genotype × environment     

Declining histone phosphorylation during myogenesis revealed by in vivo and in vitro labeling

To investigate histone phosphate levels during myogenesis, proliferation (d 1), pre-fusion postmitotic (d 2) and myotube (d 3) stage cultured chicken myoblasts were phosphorylated in vivo with [32P]orthophosphate or in vitro by incubating isolated nuclei with 32P-gamma-ATP. Incorporation of radioactive phosphate into histone was assessed by SDS and acid/urea/Triton-X-100 (AUT) gel electrophoresis and radioautography. During proliferation, in vivo labeling with [32P]orthophosphate revealed that all histones except H2b were phosphorylated in the following order of decreasing modification: H1 a greater than H2a greater than H1 b greater than H3 greater than H4. In pre-fusion post-mitotic cells phosphorylation of histones H1 a, H3 and H4 declined, whereas all histones exhibited significantly decreased modification at the myotube stage. It is unlikely that these changes resulted from decreased specific radioactivity of intracellular inorganic phosphate pools, since uptake of [32P]orthophosphate by myotubes increased six-fold, compared with proliferating cells. Isolated nuclei incubated with 32P-gamma-ATP displayed similar decreases during myogenesis; however, 1 a, H1 b and H3 were the only histones modified by in vitro phosphorylation.     

Conformational and quantitative characterization of oritavancin-peptidoglycan complexes in whole cells of Staphylococcus aureus by in vivo 13C and 15N labeling

Solid-state NMR has been used to examine the cell walls of intact whole cells of Staphyloccus aureus grown on media containing D-[1-(13)C]alanine, [(15)N]glycine, and the alanine racemase inhibitor, alaphosphin. The results of in situ site-selective, four-frequency NMR experiments show directly for the first time that (i) 54% of the cell-wall peptidoglycan stems have D-alanine termini and 46%, D-alanine-D-alanine termini; (ii) the molar ratio of stems ending in D-alanine to esterified alditol repeats of cell-wall teichoic and lipoteichoic acids is 3:2; and (iii) 50% of the mature cell-wall binding sites for a fluorinated oritavancin analogue consist of two nearest-neighbor peptide stems of different glycan strands. The drug is bound to the D-Ala-D-Ala terminus of one stem and is proximate to the bridging pentaglycyl segment that cross-links the two stems. Structural details of the binding site are revealed in a model of the glycopeptide-peptidoglycan interaction produced by molecular dynamics simulations with internuclear distance restraints determined by NMR.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.     

In vivo nitrate reductase activities in different organs of lucerne plants: Effects of combined nitrogen during first vegetative growth and after shoot harvest

Nitrate reductase (EC 1.6.6.1–3; NR) activity was evaluated in nodulated lucerne ( Medicago sativa L. cv. Europe) grown aeroponically in both the presence and absence of applied nitrogen. Determination of in vivo NR activity was done with organ pieces in 0.1 M K⁺-phosphate, pH 7.5, 0.1 M KNO₃ and 1% n -propanol. NR activity was detected in all plant parts. Leaves accounted for 40% of the whole plant activity. Root activity was as high as leaf activity. Stem NR activity accounted for 14 to 20% of the total plant activity. NR activity was also detected in symbolically dependent plants grown without combined nitrogen. Nodule NR in symbolically dependent plants accounted for 17% of the tolal plant aclivity. When nitrate was present in the nulrienl medium, NR increased 5-fold as compared lo N₂-dependenl plants. Varying levels of nitrale (1.65 to 4 m M ) had no influence on leaf or stem activities. However, root NR activity seemed to be related to the nitrale concentration in the nulrient medium. Throughoul inilial vegelative growth, in vivo NR and nitrogenase (acelylene reduction) increased simultaneously. After shoot harvest, nitrogenase (acetylene reduction) aclivity drastically decreased with reduction of photosynthate supply, whereas NR increased in all organs, especially in N₂-dependenl plants.     

Effect of source-sink relations and nitrogen nutrition on senescence and N remobilization in the flag leaf of wheat

The relation between the source-sink ratio and nitrogen nutrition on grain yield of wheat ( Triticum aestivum L. cv. Klein Chamaco) was studied in a greenhouse experiment. Plants were grown until anthesis in pots with soil fertilized with 0.16 mmol N per plant twice a week. At anthesis, all leaves but the flag leaf were excised in a group of plants. In another group the treatment consisted in a similar defoliation plus the longitudinal excision of half the ear, while a third group was left untouched as a control. At the same time, the N supply to half of the plants in each group was interrupted, while the other half continued receiving 16 m M N. The defoliated plants showed a longer functional life of the flag leaf than the control, retaining the chlorophyll, soluble proteins and total reduced nitrogen for a longer time. The ear-excised plants showed an intermediate behavior. The plants with the interrupted N supply showed a faster leaf senescence than the N supplied ones, and this coincided with an increase in the proteolytic activity and nitrogen transport to the ear. However there were no differences in ear weight between the two nitrogen treatments. It is concluded that leaves and ear compete for the nitrogen, and that a low level of carbohydrates in the flag leaf, due to a low source-sink ratio, delays leaf senescence.     

Separation of amino acid and amide nitrogen from plant extracts for 15N analysis

A rapid method was developed to obtain nitrogen for 15N analysis of individual amino acids and amides from plant tissue extracts. Amino or amide nitrogen was recovered as ammonia, suitable for preparation of samples for 15N emission spectrometry, using a combination of ion-exchange chromatography and distillation.     

The15N methodology in estimating N2 fixation by vetch and pea grown in pure stand or in mixtures with oat

Cereal-legume mixtures are frequently the best management decision for forage production instead of growing crops in pure stands. Nitrogen fertilization of cereal-legume mixtures is questionable since combined nitrogen could depress N₂ fixation by legumes. The objectives of this study were (1) to examine the effect of N fertilization on N₂ fixation by vetch and field peas in pure and in mixed stands with oats, and (2) to examine if there is any transfer of N from legumes to associated cereals. The field experiment was conducted for two growing seasons. The treatments were pure stands of vetch, pea and oats, and the mixtures of the two legumes with oats at the seeding ratios 90:10 and 75:25, fertilized with labelled¹⁵N at the rates of 15 and 90 kg N ha⁻¹. Nitrogen fertilization of 90 kg N ha⁻¹ suppressed N₂ fixation in both legumes grown in pure and in mixed stands. Crops grown in mixtures in many instances had lower atom %¹⁵N excess. Whether this was due to high N₂ fixation in the case of legume and transfer in the case of oat or the differences were due to practical problems of the¹⁵N technique is not clearly shown by the results, so based on the literature the aspect is discussed as well as the precautions which should be considered in using the¹⁵N technique in such studies.