Measurement of biological dinitrogen fixation in grassland: Comparison of the enriched 15N dilution and the natural 15N abundance methods at different nitrogen application rates and defoliation frequencies

A plant mixture of white clover (Trifolium repens L.), red clover (Trifolium pratense L.), and ryegrass (Lolium perenne L.) was established in the spring of 1991 under a cover-crop of barley. Treatments were two levels of nitrogen (400 and 20 kg N ha^-1) and two cutting intensities (3 and 6 cuts per season). Fixation of atmospheric derived nitrogen was estimated by two ¹⁵N dilution methods, one based on application of ¹⁵N to the soil, the other utilising small differences in natural abundance of ¹⁵N.Both methods showed that application of 400 kg N ha^-1 significantly reduced dinitrogen fixation, while cutting frequency had no effect. Atmospheric derived nitrogen constituted between 50 and 64% of harvested clover nitrogen in the high-N treatment, while between 73% and 96% of the harvested clover nitrogen was derived from the atmosphere in the low-N treatment. The amounts of fixed dinitrogen varied between 31–72 kg N ha^-1 and 118–161 kg N ha^-1 in the high-N and low-N treatment, respectively. The highest values for biological dinitrogen fixation were estimated by the enriched ¹⁵N dilution method.Estimates of transfer of atmospheric derived nitrogen from clover to grass obtained by the natural ¹⁵N abundance method were consistently higher than those obtained by the enriched ¹⁵N dilution method. Neither mineral nitrogen application nor defoliation frequency affected transfer of atmospheric derived nitrogen from clover to grass.Isotopic fractionation of ¹⁴N and ¹⁵N (B value) was estimated by comparing results for nitrogen fixation obtained by the enriched ¹⁵N dilution and the natural ¹⁵N abundance method, respectively. B was on average +1.20, which was in agreement with a B value determined by growing white clover in a nitrogen free media.     

Symbiotic N2-fixation by the cover crop Pueraria phaseoloides as influenced by litter mineralization

The perennial legume Pueraria phaseoloides is widely used as a cover crop in rubber and oil palm plantations. However, very little knowledge exists on the effect of litter mineralization from P. phaseoloides on its symbiotic N₂-fixation. The contribution from symbiotic N₂-fixation (Ndfa) and litter N (Ndfl) to total plant N in P. phaseoloides was determined in a pot experiment using a ¹⁵N cross-labeling technique. For determination of N₂-fixation the non-fixing plant Axonopus compressus was used as a reference. The experiment was carried out in a growth chamber during 9 weeks with a sandy soil and 4 rates of ground litter (C/N=16,2.8% N). P. phaseoloides plants supplied with the highest amount of litter produced 26% more dry matter and fixed 23% more N than plants grown in soil with no litter application, but the percentage of Ndfa decreased slightly, but significantly, from 87 to 84%. The litter N uptake was directly proportional to the rate of application and constituted 10% of total plant N at the highest application rate. Additionally, a positive correlation was found between litter N uptake and the amount of fixed N₂. The total recovery of litter N in plants averaged 26% at harvest (shoot + root) and was not affected by the quantity added. A parallel incubation experiment also showed that, as an average of all litter levels, 26% of the litter N was present in the inorganic N pool. The amounts of fertilizer and soil N taken up by plants decreased with litter application, probably due to microbial immobilization and denitrification. It is concluded that, within the litter levels studied, litter mineralization will result in a higher amount of N₂-fixed by P. phaseoloides.     

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state‐of‐the‐art quantitative 2D liquid chromatography/tandem mass spectrometry (2D‐LC‐MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size‐exclusion chromatography/inductively coupled plasma mass spectrometer (SEC‐ICP‐MS) method, in order to identify the Zn‐binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S‐transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn–nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn–nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type‐specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.     

The regulation of ammonium translocation in plants

Much controversy exists about whether or not NH(+)(4) is translocated in the xylem from roots to shoots. In this paper it is shown that such translocation can indeed take place, but that interference from other metabolites such as amino acids and amines may give rise to large uncertainties about the magnitude of xylem NH(+)(4) concentrations. Elimination of interference requires sample stabilization by, for instance, formic acid or methanol. Subsequent quantification of NH(+)(4) should be done by the OPA-fluorometric method at neutral pH with 2-mercaptoethanol as the reducing agent since this method is sensitive and reliable. Colorimetric methods based on the Berthelot reaction should never be used, as they are prone to give erroneous results. Significant concentrations of NH(+)(4), exceeding 1 mM, were measured in both xylem sap and leaf apoplastic solution of oilseed rape and tomato plants growing with NO(-)(3) as the sole N source. When NO(-)(3) was replaced by NH(+)(4), xylem sap NH(+)(4) concentrations increased with increasing external concentrations and with time of exposure to NH(+)(4). Up to 11% of the translocated N was constituted by NH(+)(4). Glutamine synthetase (GS) incorporates NH(+)(4) into glutamine, but root GS activity and expression were repressed when high levels of NH(+)(4) were supplied. Ammonium concentrations measured in xylem sap sampled just above the stem base were highly correlated with NH(+)(4) concentrations in apoplastic solution from the leaves. Young leaves tended to have higher apoplastic NH(+)(4) concentrations than older non-senescing leaves. The flux of NH(+)(4) (concentration multiplied by transpirational water flow) increased with temperature despite a decline in xylem NH(+)(4) concentration. Retrieval of leaf apoplastic NH(+)(4) involves both high and low affinity transporters in the plasma membrane of mesophyll cells. Current knowledge about these transporters and their regulation is discussed.     

Dynamic and steady-state responses of inorganic nitrogen pools and NH(3) exchange in leaves of Lolium perenne and Bromus erectus to changes in root nitrogen supply

Short- and long-term responses of inorganic N pools and plant-atmosphere NH(3) exchange to changes in external N supply were investigated in 11-week-old plants of two grass species, Lolium perenne and Bromus erectus, characteristic of N-rich and N-poor grassland ecosystems, respectively. A switch of root N source from NO(-)(3)to NH(4)(+) caused within 3 h a 3- to 6-fold increase in leaf apoplastic NH(4)(+) concentration and a simultaneous decrease in apoplastic pH of about 0.4 pH units in both species. The concentration of total extractable leaf tissue NH(4)(+) also increased two to three times within 3 h after the switch. Removal of exogenous NH(4)(+) caused the apoplastic NH(4)(+) concentration to decline back to the original level within 24 h, whereas the leaf tissue NH(4)(+)concentration decreased more slowly and did not reach the original level in 48 h. After growing for 5 weeks with a steady-state supply of NO(-)(3)or NH(4)(+), L. perenne were in all cases larger, contained more N, and utilized the absorbed N more efficiently for growth than B. erectus, whereas the two species behaved oppositely with respect to tissue concentrations of NO(-)(3), NH(4)(+), and total N. Ammonia compensation points were higher for B. erectus than for L. perenne and were in both species higher for NH(4)(+)- than for NO(-)(3)-grown plants. Steady-state levels of apoplastic NH(4)(+), tissue NH(4)(+), and NH(3) emission were significantly correlated. It is concluded that leaf apoplastic NH(4)(+) is a highly dynamic pool, closely reflecting changes in the external N supply. This rapid response may constitute a signaling system coordinating leaf N metabolism with the actual N uptake by the roots and the external N availability.     

Can silicon in glacial rock flour enhance phosphorus availability in acidic tropical soil?

Plant and Soil - Plant-available silicon (Si) is limited in strongly weathered tropical soils. The aim of the study was to evaluate Si fertilisation as a strategy to improve phosphorus (P)...     

Zinc fluxes into developing barley grains: use of stable Zn isotopes to separate root uptake from remobilization in plants with contrasting Zn status

Background and Aims

Zn imported into developing cereal grains originates from either de novo Zn uptake by the roots or remobilization of Zn from vegetative tissues. The present study was focused on revealing the quantitative importance of the two pathways for grain Zn loading and how their relative contribution varies with the overall plant Zn status.

Methods

The stable isotope ⁶⁷Zn was used to trace Zn uptake and remobilization fluxes in barley (Hordeum vulgare L.) plants growing in hydroponics at 0.1?μM (low Zn), 1.5?μM (medium Zn) or 5?μM Zn (high Zn). When grain development reached 15?days after pollination the Zn source was changed to an enriched ⁶⁷Zn isotope and plants were harvested after 6 to 48?h. Zn concentrations and isotope ratios were determined using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Results

Plants with low Zn status absorbed 3-fold more Zn than plants with medium or high Zn status when roots were exposed to an external concentration of 1.5?μM ⁶⁷Zn. Stems and ears were the primary recipients of the de novo incorporated Zn with preferential allocation to the developing grains over time. The leaves received in all cases a very small proportion (<5?%) of the newly absorbed Zn and the proportion did not increase over time. Zn fluxes derived from uptake and remobilization were almost equal in plants with low Zn status, while at high Zn status remobilization delivered 4 times more Zn to the developing grains than did root Zn uptake.

Conclusions

Stable isotopes in combination with ICP-MS provided a strong tool for quantification of Zn fluxes in intact plants. The importance of Zn remobilization compared to de novo root absorption of Zn increased with increasing plant Zn status. Very little de novo absorbed Zn was translocated to the leaves during generative growth stages.     

Leaf-atmosphere NH(3) exchange of white clover (Trifolium repens L.) in relation to mineral N nutrition and symbiotic N(2) fixation.

Plant-atmosphere NH(3) exchange was studied in white clover (Trifolium repens L. cv. Seminole) growing in nutrient solution containing 0 (N(2) based), 0.5 (low N) or 4.5 (high N) mM NO(3)(-). The aim was to show whether the NH(3) exchange potential is influenced by the proportion of N(2) fixation relative to NO(3)(-) supply. During the treatment, inhibition of N(2) fixation by NO(3)(-) was followed by in situ determination of total nitrogenase activity (TNA), and stomatal NH(3) compensation points (chi(NH(3))) were calculated on the basis of apoplastic NH4(+) concentration ([NH4(+)]) and pH. Whole-plant NH(3) exchange, transpiration and net CO(2) exchange were continuously recorded with a controlled cuvette system. Although shoot total N concentration increased with the level of mineral N application, tissue and apoplastic [NH4(+)] as well as chi(NH(3)) were equal in the three treatments. In NH(3)-free air, net NH(3) emission rates of <1 nmol m(-2) s(-1) were observed in both high-N and N(2)-based plants. When plants were supplied with air containing 40 nmol mol(-1) NH(3), the resulting net NH(3) uptake was higher in plants which acquired N exclusively from symbiotic N(2) fixation, compared to NO(3)(-) grown plants. The results indicate that symbiotic N(2) fixation and mineral N acquisition in white clover are balanced with respect to the NH4(+) pool leading to equal chi(NH(3)) in plants growing with or without NO(3)(-). At atmospheric NH(3) concentrations exceeding chi(NH(3)), the NH(3) uptake rate is controlled by the N demand of the plants.     

Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low n M range. The Mn-starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (F_v/F_m) was reduced from 0.83 in Mn-sufficient control plants to below 0.5 in Mn-starved plants. Leaf Mn concentrations declined from 30 to 7 μg Mn g⁻¹ dry weight in control and Mn-starved plants, respectively. Mn-starved plants had up to four-fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn-starved plants compared with the control. The observed increase in transpiration was substantiated by ¹³C-isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn-starved plants. The extractable wax content of leaves of Mn-starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.     

Identification and characterization of zinc-starvation-induced ZIP transporters from barley roots

Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn²⁺ transport in crop species. To expand the knowledge on Zn²⁺ transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Δzrt1/Δzrt2, defective in Zn²⁺ uptake. This strategy resulted in isolation and identification of three new Zn²⁺ transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Δzrt1/Δzrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe²⁺ and Mn²⁺ uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe²⁺ and Mn²⁺ limited media, respectively, indicating a specific role in Zn²⁺ transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in ?Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn²⁺ transporters involved in Zn²⁺ homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.