Anthony D. M. Glass

Molecular and physiological aspects of nitrate uptake in plants

Abstract Nitrate is an important macronutrient and also acts as a signal for plant growth; however, its levels in the soil solution can vary by three to four orders of magnitude. Consequently, plants have evolved regulated, energy dependent systems for the uptake of nitrate using both high and low affinity transporters. Genes that encode representatives of each class of transport system have been identified and fall into two families: NRT1 and NRT2 . Members of these families are induced in response to nitrate in the environment and are regulated by internal signals including nitrogen metabolites and shoot demand for nitrogen. The evidence to date indicates that the NRT2 transporters contribute specifically to the nitrate-inducible, high affinity nitrate uptake system while the NRT1 transporters contribute more broadly to nitrogen uptake and show both inducible and constitutive expression.

Utilization index: A modified approach to the estimation and comparison of nutrient utilization efficiency in plants

Abstract The disadvantages of using utilization quotient (biomass per unit amount of nutrient present in biomass) in comparing nutrient utilization efficiencies of different varieties and species are discussed. A modified approach to the estimation of utilization efficiency is presented. A comparison of efficiencies of two plants calculated by this method gives an index which is the ratio of biomass ratio: tissue nutrient concentration ratio. Theoretical validity and advantages in practical application of this approach are discussed.

Nitrogen Use Efficiency of Crop Plants: Physiological Constraints upon Nitrogen Absorption

Current global nitrogen fertilizer use has reached approximately one hundred billion kg per annum. In many agricultural systems, a very substantial portion of this applied nitrogen fertilizer is lost from soil to groundwaters, rivers and oceans. While soil physicochemical properties play a significant part in these losses, there are several characteristic features of plant nitrogen transporter function that facilitate N losses. Nitrate and ammonium efflux from roots result in a reduction of net nitrogen uptake. As external nitrate and ammonium concentrations, respectively, are increased, particularly into the range of concentrations that are typical of agricultural soils, elevated rates of nitrate and ammonium efflux result. The rapid down-regulation of high-affinity influx as plants become nitrogen replete further reduces the roots capacity to acquire external nitrogen; only nitrogen-starved roots absorb with both high capacity and high affinity. The results of studies using molecular biology methods demonstrate that genes encoding nitrate and ammonium transporters are rapidly down-regulated when nitrogen is resupplied to nitrogen-starved plants. Provision of ammonium to roots of plants actively absorbing nitrate imposes a block on nitrate uptake, the extent of which depends on the ammonium concentration, thus further reducing the efficient utilization of soil nitrate. During the daily variation of incoming light and during periods of low incident irradiation (i.e. heavy cloud cover) the expression levels of genes encoding nitrate and ammonium transporters, and rates of nitrate and ammonium uptake, are substantially reduced. Low temperatures reduce growth and nitrogen demand, and appear to discriminate against high-affinity nitrogen influx. In sum, these several factors conspire to limit rates of plant nitrogen uptake to values that are well below capacity. These characteristics of the plants nitrogen uptake systems facilitate nitrogen losses from soils.

Dissection of the AtNRT2.1:AtNRT2.2 Inducible High-Affinity Nitrate Transporter Gene Cluster

Using a new Arabidopsis (Arabidopsis thaliana) mutant (Atnrt2.1-nrt2.2) we confirm that concomitant disruption of NRT2.1 and NRT2.2 reduces inducible high-affinity transport system (IHATS) by up to 80%, whereas the constitutive high-affinity transport system (CHATS) was reduced by 30%. Nitrate influx via the low-affinity transport system (LATS) was unaffected. Shoot-to-root ratios were significantly reduced compared to wild-type plants, the major effect being upon shoot growth. In another mutant uniquely disrupted in NRT2.1 (Atnrt2.1), IHATS was reduced by up to 72%, whereas neither the CHATS nor the LATS fluxes were significantly reduced. Disruption of NRT2.1 in Atnrt2.1 caused a consistent and significant reduction of shoot-to-root ratios. IHATS influx and shoot-to-root ratios were restored to wild-type values when Atnrt2.1-nrt2.2 was transformed with a NRT2.1 cDNA isolated from Arabidopsis. Disruption of NRT2.2 in Atnrt2.2 reduced IHATS by 19% and this reduction was statistically significant only at 6 h after resupply of nitrate to nitrogen-deprived plants. Atnrt2.2 showed no significant reduction of CHATS, LATS, or shoot-to-root ratios. These results define NRT2.1 as the major contributor to IHATS. Nevertheless, when maintained on agar containing 0.25 mm KNO3 as the sole nitrogen source, Atnrt2.1-nrt2.2 consistently exhibited greater stress and growth reduction than Atnrt2.1. Evidence from real-time PCR revealed that NRT2.2 transcript abundance was increased almost 3-fold in Atnrt2.1. These findings suggest that NRT2.2 normally makes only a small contribution to IHATS, but when NRT2.1 is lost, this contribution increases, resulting in a partial compensation.

High-Affinity Nitrate Transport in Roots of Arabidopsis Depends on Expression of the NAR2-Like Gene AtNRT3.1

The NAR2 protein of Chlamydomonas reinhardtii has no known transport activity yet it is required for high-affinity nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the nitrate transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO3 after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.

Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage

AbstractThe NRT2.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-β-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips. Abbreviation: GUS, β-glucuronidase

Nitrogen transport in plants, with an emphasis on the regulation of fluxes to match plant demand

Physiological methods, especially the use of isotopes of N, have allowed for the detailed characterizations of the several putative transport systems for nitrate and ammonium in roots of higher plants. In the last decade, the cloning of genes that appear to encode both high- and low-affinity transporters represent major advances, as well as substantiating the inferences based on earlier physiological methods. Nevertheless, the unexpected plethora of genes that have been identified now presents even greater challenges, to resolve their individual functions and to attempt to place these functions in a whole plant/environmental context.

The Effects of Aluminum on the Influx of Calcium, Potassium, Ammonium, Nitrate, and Phosphate in an Aluminum-Sensitive Cultivar of Barley (Hordeum vulgare L.).

The mechanism by which aluminum interferes with ion influx is not known. In this study, the effects of aluminum on the influx of the cations calcium, potassium, and ammonium and the anions nitrate and phosphate were measured in an aluminum-sensitive cultivar of barley (Hordeum vulgare L.). Aluminum (100 [mu]M) was found to inhibit the influx of the cations calcium (69%), ammonium (40%), and potassium (13%) and enhancing the influx of the anions nitrate (44%) and phosphate (17%). Aluminum interfered with the binding of the cations in the cell wall by the same order of magnitude as their respective influxes, whereas phosphate binding was strongly enhanced. The results are consistent with a mechanism whereby aluminum binds to the plasma membrane phospholipids, forming a positively charged layer that influences ion movement to the binding sites of the transport proteins. A positive charge layer would retard the movement of cations and increase the movement of anions to the plasma membrane in proportion to the charges carried by these ions.

Functional Analysis of an Arabidopsis T-DNA “Knockout” of the High-Affinity NH4 + Transporter AtAMT1;1

NH4 + acquisition by plant roots is thought to involve members of the NH4 +transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH4 +. Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH4 + influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity 13NH4 +influx in the amt1;1:T-DNAmutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity 13NH4 +influx in the mutant by 30% compared with wild-type plants, whereas low-affinity 13NH4 + influx (250 μm–10 mm NH4 +) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 andAtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNAplants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH4 + and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH4 + transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.

Ammonium Inhibition of Arabidopsis Root Growth Can Be Reversed by Potassium and by Auxin Resistance Mutations aux1, axr1, and axr2

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3–12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20–)100 [mu]M) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.