Enhancement of cadmium accumulation in sweet sorghum as affected by nitrate

• The Cadmium (Cd)‐polluted soils are is an increasing concern worldwide. Phytoextraction of Cd pollutants by high biomass plants, such as sweet sorghum, is considered an environmentally‐friendly, cost‐effective and sustainable strategy for remediating this problem. Nitrogen (N) is a macronutrient essential for plant growth, development and stress resistance. Nevertheless, how nitrate, as an important form of N, affects Cd uptake, translocation and accumulation in sweet sorghum is still unclear.
• In the present study, a series of nitrate levels (N1, 0.5 mm ; N2, 2 mm ; N3, 4 mm ; N4, 8 mm and N5, 16 mm ) with or without added 5 μm CdCl₂ treatment in sweet sorghum was investigated hydroponically.
• The results indicate that Cd accumulation in the aboveground parts of sweet sorghum was enhanced by optimum nitrate supply, resulting from both increased dry weight and Cd concentration. Although root‐to‐shoot Cd translocation was not enhanced by increased nitrate, some Cd was transferred from cell walls to vacuoles in leaves. Intriguingly, expression levels of Cd uptake and transport genes, SbNramp1, SbNramp5 and SbHMA3, were not closely related to increased Cd as affected by nitrate supply. The expression of SbNRT1.1B in relation to nitrate transport showed an inverted ‘U’ shape with increasing nitrate levels under Cd stress, which was in agreement with trends in Cd concentration changes in aboveground tissues.
• Based on the aforementioned results, nitrate might regulate Cd uptake and accumulation through expression of SbNRT1.1B rather than SbNramp1, SbNramp5 or SbHMA3, the well‐documented genes related to Cd uptake and transport in sweet sorghum.

     

Associative bacteria influence maize (Zea mays L.) growth,physiology and root anatomy under different nitrogen levels

• Despite the great diversity of plant growth‐promoting bacteria (PGPB ) with potential to partially replace the use of N fertilisers in agriculture, few PGPB have been explored for the production of commercial inoculants, reinforcing the importance of identifying positive plant–bacteria interactions. Aiming to better understand the influence of PGPB inoculation in plant development, two PGPB species with distant phylogenetic relationship were inoculated in maize.
• Maize seeds were inoculated with Bacillus sp. or Azospirillum brasilense . After germination, the plants were subjected to two N treatments: full (N+) and limiting (N?) N supply. Then, anatomical, biometric and physiological analyses were performed.
• Both PGPB species modified the anatomical pattern of roots, as verified by the higher metaxylem vessel element (MVE ) number. Bacillus sp. also increased the MVE area in maize roots. Under N+ conditions, both PGPB decreased leaf protein content and led to development of shorter roots; however, Bacillus sp. increased root and shoot dry weight, whereas A. brasilense increased photosynthesis rate and leaf nitrate content. In plants subjected to N limitation (N?), photosynthesis rate and photosystem II efficiency increased in maize inoculated with Bacillus sp., whilst A. brasilense contained higher ammonium, amino acids and total soluble sugars in leaves, compared to the control.
• Plant developmental and metabolical patterns were switched by the inoculation, regardless of the inoculant bacterium used, producing similar as well as distinct modifications to the parameters studied. These results indicate that even non‐diazotrophic inoculant strains can improve the plant N status as result of the morpho‐anatomical and physiological modifications produced by the PGPB .

     

The influence of nitrate supply on nitrogen fixation during growth of the field bean Vicia faba in sand

Field bean (Vicia faba L.) cv. Maris Bead seeds were inoculated with Rhizobium Catalogue No. 1001, supplied by Rothamsted Experimental Station and grown in sand culture supplied with ¹⁵N-labelled nitrate at two concentrations. Plants were sampled at intervals throughout their growth for ¹⁵N and total N analysis. The rate of nitrate uptake was almost uniform up to pod-fill and was proportional to the nitrate concentration. Nodule weight was slightly depressed by the larger nitrate concentration at all samplings, and there was a corresponding reduction in the amount of atmospheric nitrogen fixed. However, at harvest the bean seeds from plants given most nitrate contained slightly more total N, as the enhanced nitrate uptake outweighed the reduction in fixation.     

Sink-stimulated photosynthesis and sink-dependent increase in nitrate uptake: nitrogen and carbon relations of the parasitic association Cuscuta reflexa–Ricinus communis

Ricinus communis L. was grown under limiting N supply in quartz sand culture, fed with 0.2, 1 or 5 mol m^?3 NO₃^?, or in liquid culture with 0.022, 0.05 or 0.5 mol m^?3 NO₃^?. Some of the plants were infected with Cuscuta reflexa Roxb. As occurred for the host, dry matter production and growth of C. reflexa were severely depressed with decreasing N supply to the host. When parasitized by C. reflexa, the shoot and root dry weight of Ricinus was diminished at all levels of N nutrition, but the total dry weight of host plus parasite was almost the same as that of uninfected Ricinus. In contrast to the situation in Lupinus albus (Jeschke et al. 1994b), infection by Cuscuta resulted in increased tissue N levels in the host and the N content of the system Ricinus plus C. reflexa was the same or even somewhat larger than that of uninfected plants. This indicated a sink-dependent stimulation of nitrate uptake. As a result of decreased root weights, nitrate uptake g^?1 FW was stimulated by 80, 60 or only 40% at 0.2, 1 or 5 mol m^?3 nitrate supply. Increased nitrate uptake was reflected, particularly at low N supply, in xylem transport; xylem sap nitrate concentrations were substantially elevated, while those of amino acids were decreased in parasitized plants. This indicated an inhibition of nitrate assimilation in roots of parasitized plants under limiting N supply. Besides these effects on N relations, C. reflexa induced a substantial sink-dependent stimulation of net photosynthesis in host leaves and a concomitant increase in stomatal opening and transpiration. This stimulation depended on the relative sink size induced by Cuscuta, on nitrogen nutrition and on leaf age, indicating that delayed senescence of leaves contributes to the overall effects of Cuscuta on its host. The Cuscuta-induced inhibition of nitrate assimilation in the roots and the increase in nitrate uptake suggest that nitrate reduction was shifted towards the leaves in the presence of C. reflexa. The stimulating effects of C. reflexa in the Ricinus-Cuscuta association are compared with the strongly inhibitory effects occurring in the tripartite association L. albus–Rhizobium–Cuscuta reflexa.     

The response ofPlantago lanceolata L. andP. major L. spp.major to nitrate depletion

To study aspects of the ecology of grassland species, in a comparative experiment, plants ofP. lanceolata andP. major were grown in pots in a greenhouse, and subjected to a gradual nitrate depletion for several weeks. Control plants were weekly supplied with nitrate. Growth, leaf appearance and disappearance, concentrations of cations and inorganic anions, soluble and insoluble reduced nitrogen concentrations,in vivo nitrate reductase activity (NRA) and the concentration of non-structural carbohydrates in several parts of the plants were followed. Depletion of nitrate caused a reduction of shoot growth, both in biomass and number of leaves. Withering of leaves increased. Accumulation of root dry matter was little (P. lanceolata), or not (P. major) affected. The concentration of reduced nitrogen in all tissues also decreased, both that of the soluble and that of the insoluble fraction. As a result, nitrogen use efficiency (NUE, g dry matter produced per mmol N incorporated) increased by nitrate depletion. NRA was higher in the roots than in the leaves, and decreased with increasing nitrate depletion. In control plants, nitrate became also limiting. This resulted in decreasing nitrate concentrations in leaves and roots. In the leaves, the decrease in nitrate concentration was preceded by a decrease in NRA. The decrease of the nitrate concentration was parallelled by an increase in the concentration of soluble sugar. No major differences in the response towards nitrate depletion were observed between the two species. Grassland Species Research Group, publication no. 129     

Tight control of nitrate acquisition in a plant species that evolved in an extremely phosphorus‐impoverished environment

Hakea prostrata (Proteaceae) has evolved in an extremely phosphorus (P)‐limited environment. This species exhibits an exceptionally low ribosomal RNA (rRNA) and low protein and nitrogen (N) concentration in its leaves. Little is known about the N requirement of this species and its link to P metabolism, despite this being the key to understanding how it functions with a minimal P budget. H. prostrata plants were grown with various N supplies. Metabolite and elemental analyses were performed to determine its N requirement. H. prostrata maintained its organ N content and concentration at a set point, independent of a 25‐fold difference nitrate supplies. This is in sharp contrast to plants that are typically studied, which take up and store excess nitrate. Plants grown without nitrate had lower leaf chlorophyll and carotenoid concentrations, indicating N deficiency. However, H. prostrata plants at low or high nitrate availability had the same photosynthetic pigment levels and hence were not physiologically compromised by the treatments. The tight control of nitrate acquisition in H. prostrata retains protein at a very low level, which results in a low demand for rRNA and P. We surmise that the constrained nitrate acquisition is an adaptation to severely P‐impoverished soils.     

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Effects of rhizospheric bicarbonate on net nitrate uptake and partitioning between the main nitrate utilising processes in Populus canescens and Sambucus nigra

Increased levels of rhizospheric dissolved inorganic carbon have repeatedly been demonstrated to enhance plant growth by up to 80%, although carbon from dark fixation accounts for only 1–3% of total plant carbon gain. This study, therefore, aimed at investigating the effects of bicarbonate on nitrate uptake, assimilation and translocation to shoots. Clonal saplings of poplar (Populus canescens(Ait.) Sm.) and elder (Sambucus nigraL.) were grown hydroponically for 35 days in a nutrient solution containing 0, 0.5 and 1 mM bicarbonate and 2 mM nitrate as the sole nitrogen source at pH 7.0. Net nitrate uptake, root nitrate accumulation and reduction, and export of nitrogenous solutes to shoots were measured after incubating plants with ¹⁵N-labelled nitrate for 24 h. Net nitrate uptake increased non-significantly in plant species (19–61% compared to control plants) in response to 1 mM bicarbonate. Root nitrate reduction and nitrogen export to shoots increased by 80 and 95% and 15 and 44% in poplar and elder, respectively. With enhanced root zone bicarbonate, both species also exhibited a marked shift between the main nitrate utilising processes. Poplar plants increasingly utilised nitrate via nitrate reduction (73–88% of net nitrate uptake), whereas the proportions of export (20–9%) and storage in roots (7–3%) declined as plants were exposed to 1 mM external bicarbonate. On the other hand, elder plants exhibited a significant increase of root nitrate reduction (44–66%) and root nitrate accumulation (6–25%). Nitrate translocation to elder shoots decreased from 50 to 8% of net nitrate uptake. The improved supply of nitrogen to shoots did not translate into a significant stimulation of growth, relative growth rates increased by only 16% in poplar saplings and by 7% in elder plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Responses of leaf C:N:P stoichiometry to water supply in the desert shrub Zygophyllum xanthoxylum

• Based on the elemental composition of major biochemical molecules associated with different biological functions, the ‘growth rate hypothesis’ proposed that organisms with a higher growth rate would be coupled to lower C:N, especially lower C:P and N:P ratios. However, the applicability of the growth rate hypothesis for plants is unclear, especially for shrubs growing under different water supply.
• We performed an experiment with eight soil moisture levels (soil water content: 4%, 6%, 8%, 13%, 18%, 23%, 26% and 28%) to evaluate the effects of water availability on leaf C:N:P stoichiometry in the shrub Zygophyllum xanthoxylum.
• We found that leaves grew slowly and favored accumulation of P over C and N under both high and low water supply. Thus, leaf C:P and N:P ratios were unimodally related to soil water content, in parallel with individual leaf area and mass. As a result, there were significant positive correlations between leaf C:P and N:P with leaf growth (u).
• Our result that slower‐growing leaves had lower C:P and N:P ratios does not support the growth rate hypothesis, which predicted a negative association of N:P ratio with growth rate, but it is consistent with recent theoretical derivations of growth–stoichiometry relations in plants, where N:P ratio is predicted to increase with increasing growth for very low growth rates, suggesting leaf growth limitation by C and N rather than P for drought and water saturation.

     

Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs

We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.Abbreviations DAP Days after planting - Gln Glutamine - NR Nitrate reductase - WT Wild type     

Physiological dissection revealed that both uptake and assimilation are the major components regulating different growth responses of two tobacco cultivars to nitrogen nutrition

• K326 and HD represent major tobacco cultivars in China, which required large N fertiliser input but at different application rates. To understand primary components affecting tobacco N use physiology, we adopted these two varieties as valuable genetic material to assess their growth response to N nutrition.
• We established a hydroponic culture system to grow plants supplied with different N regimes. Plant biomass, N, ammonium, nitrate, arginine, GS and NR activity, N transfer and use efficiency as well as root uptake were examined.
• Our data revealed the preference of K326 and HD to utilise nitrate or ammonium nitrate but not ammonium alone, with 2 mm N supply probably sufficient and economical to achieve good biomass production at the vegetative stage. Moreover, both varieties were very sensitive to ammonium, perhaps due to lack of or abnormal signalling related to nitrate and/or arginine rather than impairment of N acquisition and initial assimilation; this was supported by measurements of the plant content of N, ammonium and activities of GS and NR. Notably, short‐term ¹⁵N root influx studies identified differential uptake kinetics of K326 and HD, with distinct affinities and transport rates for ammonium and nitrate.
• The data suggest that the growth adaptation of K326 or HD to higher or lower N may be ascribed to different competences for effective N uptake/translocation and assimilation. Thus, our work provides valuable information to prompt deeper investigation of the molecular basis controlling plant N use efficiency.

     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Relationship of nitrate supply with growth rate, plasma membrane-bound and cytosolic nitrate reductase, and tissue nitrate content in tobacco plants

cNR, cytosolic nitrate reductase
PM-NR, plasma membrane-bound nitrate reductase

Activities of plasma membrane-bound nitrate reductase (PM-NR) and cytosolic nitrate reductase (cNR) in tobacco (Nicotiana tabacum L. cv. Samsun) are regulated differently, depending upon the nitrate supply to the culture medium (in sand culture). The cNR activity of roots was higher at low nitrate concentrations with the maximum at 5 mM nitrate supply and declined to low values beyond 5 mM . In contrast, the PM-NR activity of roots increased with higher nitrate concentrations with the maximum at 25 mM nitrate and clearly decreased only at 40 mM . This high PM-NR activity correlated with a low growth rate and might be one of the responses to excess nitrate. Internal nitrate and total nitrogen content of the tissues, however, showed a relative minimum in shoots and in roots of between 15 and 25 mM external nitrate. With declining PM-NR activities beyond 25 mM external nitrate, the nitrate content in the tissue increased indicating an inverse relationship between tissue nitrate content and root PM-NR activity. In leaves both NR activities (cNR and PM-NR) correlated with the internal nitrate content, but with a different response at low nitrate.     

Elevated pCO2 Affects C and N Metabolism in Wild Type and Transgenic Tobacco Exhibiting Altered C/N Balance in Metabolite Analysis

Abstract: Diurnal changes in starch, sugar and amino acid concentrations in source leaves, sink leaves and roots of tobacco plants were determined. In addition to wild type tobacco, transformed plants deficient in root nitrate reductase and exhibiting decreased rates of growth were employed. Further, the growth rates of tobacco plants were modulated by exposure to elevated pCO₂. From the diurnal alterations in metabolite concentrations, the daily turnover of starch and amino N was estimated in order to: (i) elucidate whether turnover rates can be related to growth rates, and (ii) identify individual amino compounds with the potential to indicate nitrogen fluxes and the C/N status of plants. Elevated pCO₂ increased growth rates and daily turnover of starch in both wild type and transformed plants, indicating enhanced rates of photosynthesis. In wild type plants, elevated pCO₂ increased the turnover of amino N, notably glutamine and alanine, in mature source leaves, indicating enhanced nitrate reduction. By contrast, amino N turnover in source leaves of transformed plants was not affected by elevated pCO₂, although nitrate reduction was presumably enhanced. Apparently, export of amino N was increased from the source leaves of transformed plants. This assumption was supported by a significantly increased turnover of amino N in young sink leaves compared to mature source leaves, indicating a preference for acropetal amino N allocation and import into the young leaves of the transformed plants. Further, elevated pCO₂ increased the allocation of leaf‐derived amino N to the roots of transformed plants. This led to increased levels of amino compounds during the entire day, notably glutamate, but did not affect root growth of the transformed plants. The suitability of individual amino compounds as markers for major N fluxes, such as nitrate reduction, photorespiration, and amino N export and import is discussed.     

Response of mesquite to nitrate and salinity in a simulated phreatic environment: Water use,dry matter and mineral nutrient accumulation

Mesquite plants (Prosopis glandulosa var. Torreyana) were grown in 2-m long columns 20 cm in diameter, and provided with a constant, stable ground water source 10 cm above the sealed base of the column. Ground water contained 0, 1 or 5 mM nitrate, or a mixed salt solution (1.4, 2.8, or 5.6 dS m^-1) with the ionic ratios of ground water found in a field stand of Prosopis at Harper's Well (2.8 dS m^-1). Water uptake in the highly salinized columns began to decrease relative to low salt columns when soil salinity probes 30 cm above the column base read approximately 28 dS m^-1, a potential threshold for mesquite salt tolerance. Prosopis growth increased with increasing nitrate, and decreased with increasing salinity. Water use efficiency was little affected by treatment, averaging approximately 2 g dry matter L^-1 water used. Most fine roots were recovered from a zone about 25 cm above the ground water surface where water content and aeration appeared to be optimal for root growth. Root-shoot ratio was little affected by nitrate, but increased slightly with increasing salinity. Plant tissue P concentrations tended to increase with increasing salinity and decrease with increasing N, approaching potentially deficient foliage concentrations at 5 mM nitrate. The whole-plant leaf samples increased in sodium concentration both with added salt and with added nitrate. Foliar manganese concentrations increased with increasing salt in the absence of nitrate. Concentrations of sodium in leaves were low (<10 g kg^-1), considering the high salt concentrations in the ground water. Prosopis appears to exclude sodium very effectively, especially from its younger leaves. Although Prosopis is highly salt tolerant, the degree to which it utilizes soil nitrate in place of biologically fixed N may lower its salinity tolerance and affect its nutrient relations in phreatic environments.     

Fate of nitrate during initial nitrate utilization by nitrogen-depleted dwarf bean

The fate of nitrate and nitrogen-15 was followed during the apparent induction phase (6h) for nitrate uptake by N-depleted dwarf bean (Phaseolus vulgaris L. ev. Witte Krombek). Experiments were done with intact plants and with detached root systems. Qualitatively and quantitatively, xylem exudation from detached roots was a bad estimate of the export of NO^?₃ or NO^?₃-¹⁵N from roots of intact plants. In vivo nitrate reductase activity (NRA) agreed well with in situ reduction, calculated as the difference between uptake and accumulation in whole plants, provided NRA was assayed with merely endogenous nitrate as substrate (‘actual’ NRA). The majority (75%) of the entering nitrate remained unmetabolized. Both nitrate reduction and nitrate accumulation occurred predominantly in the root system. Some (< 25%) of the root-reduced nitrate-N was translocated to the shoot. Nitrate uptake occurred against the concentration gradient between medium and root cells, and probably against the gradient of the electro-chemical potential of nitrate. Part of the energy expended for NO^?₃ absorption came from the tops, since decapitation and ringing at the stem base restricted nitrate uptake.