Trevor Garnett

Root based approaches to improving nitrogen use efficiency in plants

In the majority of agricultural growing regions, crop production is highly dependent on the supply of exogenous nitrogen (N) fertilizers. Traditionally, this dependency and the use of N-fertilizers to restore N depleted soils has been rewarded with increased plant health and yields. In recent years, increased competition for non-renewable fossil fuel reserves has directly elevated prices of N-fertilizers and the cost of agricultural production worldwide. Furthermore, N-fertilizer based pollution is becoming a serious issue for many regions where agriculture is highly concentrated. To help minimize the N footprint associated with agricultural production there is significant interest at the plant level to develop technologies which can allow economically viable production while using less applied N. To complement recent reviews examining N utilization efficiency in agricultural plants, this review will explore those strategies operating specifically at the root level, which may directly contribute to improved N use efficiencies in agricultural crops such as cereals, where the majority of N-fertilizers are used and lost to the environment. Root specific phenotypes that will be addressed in the context of improvements to N acquisition and assimilation efficiencies include: root morphology; root to shoot ratios; root vigour, root length density; and root N transport and metabolism.

Dichotomy in the NRT Gene Families of Dicots and Grass Species

A large proportion of the nitrate (NO3 −) acquired by plants from soil is actively transported via members of the NRT families of NO3 − transporters. In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes (maize, rice, sorghum, Brachypodium). We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO3 − transporters and NO3 − transport in grass crop species.

Nutritional and chlorophyll fluorescence responses of lucerne (Medicago sativa) to waterlogging and subsequent recovery

Periodic flooding of perennial crops such as lucerne (Medicago sativa,L) is a major cause of lowered productivity and leads in extreme cases to plant death. In this study, effects of waterlogging and subsequent recovery on plant nutrient composition and PSII photochemistry were studied to gain a better understanding of the mechanisms of recovery as they relate to leaf photochemistry (chlorophyll fluorescence) and nutrient dynamics. Three lucerne cultivars and one breeding line were flooded for 20 d, drained and left to recover for another 16 d under glasshouse conditions. Leaf and root nutrient composition (P, K, Ca, Mg, B, Cu and Zn) of waterlogged lucerne was significantly lower than in freely drained controls, leaf N concentrations were also significantly lower in waterlogged lucerne. At the same time, there were significantly (5-fold) higher concentrations of Fe in waterlogged roots and Na in leaves (2-fold) of stressed plants. PS II photochemistry, which was impaired due to waterlogging, recovered almost fully after 16 d of free drainage in all genotypes. Alongside fluorescence recovery, concentrations of several nutrients also increased in recovered plants. Growth parameters, however, remained suppressed after draining. The latter was due to both the smaller capacity of CO2 assimilation in previously waterlogged plants (caused in part by nutrient deficiency and associated inhibition of PSII) and the plant’s need to re-direct available nutrient and assimilate pools to repair the damage to the photosynthetic apparatus and roots. It is concluded, that for any lucerne-breeding program it is important to determine not only the degree of tolerance to waterlogging but also the potential for recovery of different genotypes, as well as look for ‘outstanding individuals’ within each population.

Multiple traits associated with salt tolerance in lucerne: revealing the underlying cellular mechanisms

Salinity tolerance is a complex trait inferring the orchestrated regulation of a large number of physiological and biochemical processes at various levels of plant structural organisation. It remains to be answered which mechanisms and processes are crucial for salt tolerance in lucerne (Medicago sativa L.). In this study, salinity effects on plant growth characteristics, pigment and nutrient composition, PSII photochemistry, leaf sap osmolality, changes in anatomical and electrophysiological characteristics of leaf mesophyll, and net ion fluxes in roots of several lucerne genotypes were analysed. Salinity levels ranged from 40 to ~200 mm NaCl, and were applied to either 2-month-old plants or to germinating seedlings for a period of between 4 and 12 weeks in a series of hydroponic, pot and field experiments. Overall, the results suggest that different lucerne genotypes employ at least two different mechanisms for salt tolerance. Sodium exclusion appeared to be the mechanism employed by at least one of the tolerant genotypes (Ameristand 801S). This cultivar had the lowest leaf thickness, as well as the lowest concentration of Na+ in the leaf tissue. The other tolerant genotype, L33, had much thicker leaves and almost twice the leaf Na+ concentration of Ameristand. Both cultivars showed much less depolarisation of leaf membrane potential than the sensitive cultivars and, thus, had better K+ retention ability in both root and leaf tissues. The implications of the above measurements for screening lucerne germplasm for salt tolerance are discussed.

Ammonium and nitrate uptake by Eucalyptus nitens: effects of pH and temperature

Ammonium and nitrate uptake by roots of Eucalyptus nitens was characterised with respect to pH and temperature. Uptake of ammonium and nitrate was measured as depletion from solutions by roots of intact 11 week old solution-cultured seedlings. Uptake rates of ammonium were consistently higher than those of nitrate in all experiments. Uptake rates for ammonium were 200% higher at pH 4 than at pH 6, but for nitrate were unchanged. Uptake rates of ammonium and nitrate were both reduced to a similar extent (70%) with a decrease in temperature from 20 °C to 10 °C. For ammonium uptake, there was rapid (<24 hr) adaptation to a reduction in root temperature. The apparent preference shown here for ammonium over nitrate could be indicative of E. nitens growing in cold, acidic forest soils where ammonium is commonly more available than nitrate. These results suggest that N uptake rates of E. nitens may be maximised under a wide variety of conditions if N is supplied predominantly in the ammonium form.

The response of the maize nitrate transport system to nitrogen demand and supply across the lifecycle

An understanding of nitrate (NO3-) uptake throughout the lifecycle of plants, and how this process responds to nitrogen (N) availability, is an important step towards the development of plants with improved nitrogen use efficiency (NUE). NO3- uptake capacity and transcript levels of putative high- and low-affinity NO3- transporters (NRTs) were profiled across the lifecycle of dwarf maize (Zea mays) plants grown at reduced and adequate NO3-. Plants showed major changes in high-affinity NO3- uptake capacity across the lifecycle, which varied with changing relative growth rates of roots and shoots. Transcript abundances of putative high-affinity NRTs (predominantly ZmNRT2.1 and ZmNRT2.2) were correlated with two distinct peaks in high-affinity root NO3- uptake capacity and also N availability. The reduction in NO3- supply during the lifecycle led to a dramatic increase in NO3- uptake capacity, which preceded changes in transcript levels of NRTs, suggesting a model with short-term post-translational regulation and longer term transcriptional regulation of NO3- uptake capacity. These observations offer new insight into the control of NO3- uptake by both plant developmental processes and N availability, and identify key control points that may be targeted by future plant improvement programmes to enhance N uptake relative to availability and/or demand.

Rice DUR3 mediates high-affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis

• Despite the great agricultural and ecological importance of efficient use of urea-containing nitrogen fertilizers by crops, molecular and physiological identities of urea transport in higher plants have been investigated only in Arabidopsis. • We performed short-time urea-influx assays which have identified a low-affinity and high-affinity (K(m) of 7.55 μM) transport system for urea-uptake by rice roots (Oryza sativa). • A high-affinity urea transporter OsDUR3 from rice was functionally characterized here for the first time among crops. OsDUR3 encodes an integral membrane-protein with 721 amino acid residues and 15 predicted transmembrane domains. Heterologous expression demonstrated that OsDUR3 restored yeast dur3-mutant growth on urea and facilitated urea import with a K(m) of c. 10 μM in Xenopus oocytes. • Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis revealed upregulation of OsDUR3 in rice roots under nitrogen-deficiency and urea-resupply after nitrogen-starvation. Importantly, overexpression of OsDUR3 complemented the Arabidopsis atdur3-1 mutant, improving growth on low urea and increasing root urea-uptake markedly. Together with its plasma membrane localization detected by green fluorescent protein (GFP)-tagging and with findings that disruption of OsDUR3 by T-DNA reduces rice growth on urea and urea uptake, we suggest that OsDUR3 is an active urea transporter that plays a significant role in effective urea acquisition and utilisation in rice.

Simultaneous measurement of ammonium, nitrate and proton fluxes along the length of eucalypt roots

Knowledge of the preferred source of N for Eucalyptus nitens will lead to improved fertiliser management practices in plantations. Ion selective microelectrodes were used non-invasively to measure simultaneously net fluxes of NH4+, NO3− and H+ along the tap root of solution-cultured E. nitens. Measurements were conducted in solutions containing 100 μm NH4NO3. The pattern of fluxes was such that there was a large influx of NH4+, a smaller influx of NO3− and large H+ efflux. The ratio of these fluxes was constant, according to the ratio 3:1:−6 (NH4+:NO3−:H+). Within the region 20–60 mm from the root apex of E. nitens seedlings there was spatial and temporal variation in fluxes but flux patterns remained constant. Root hair density did not affect fluxes nor did proximity to lateral roots. Variation was less than that found in previous studies of localised root fluxes using similar high-resolution measurement techniques. It was concluded that small-scale spatial variation in fluxes may have confounded previous studies. There were associations between fluxes of all three ions, the strongest associations being between NH4+ and H+, and NH4+ and NO3−. Overall, these results are consistent with NH4+ being the preferred source N for E. nitens.

Kinetics of ammonium and nitrate uptake by eucalypt roots and associated proton fluxes measured using ion selective microelectrodes

Ion-selective microelectrodes were used non-invasively to measure the concentration dependence of NH4+ and NO3- fluxes around the roots of intact solution-cultured Eucalyptus nitens (Deane & Maiden) Maiden. In addition, NH4+ and H+ fluxes were measured simultaneously at a range of NH4+ concentrations, and NO3- and H+ fluxes were measured simultaneously at a range of NO3- concentrations. Nitrogen concentrations ranged from 10-250 μM, i.e. in the range corresponding to the high affinity transport system (HATS). Both NH4+ and NO3- fluxes exhibited saturating Michaelis-Menten-style kinetics. The Km was 16 μM for NH4+ and 18 μM for NO3-. Values of Vmax were 53 nmol m-2 s-1 for NH4+ and 37 nmol m-2 s-1 for NO3-. Proton fluxes were highly correlated with NH4+ and NO3- fluxes, but the relationships were different. Proton efflux increased with increasing NH4+ concentration and mirrored the changing NH4+ fluxes. The ratio between NH4+ and H+ fluxes was 1 : -1.6. Proton influx was evident with initial exposure to NO3-, with the flux stoichiometry for NO3- : H+ being 1 : 1.4. Subsequent increases in NO3- concentration caused a gradual increase in H+ efflux such that the flux stoichiometry for NO3- : H+ became 1 : -0.8. The presence of 100 μM NH4+ greatly reduced NO3- fluxes and caused a large and constant H+ efflux. These results are evidence that E. nitens has a preference for NH4+ as a source of N, and that the fluxes of NH4+ and NO3- are quantitatively linked to H+ flux.

Genetic approaches to enhancing nitrogen-use efficiency (NUE) in cereals: challenges and future directions

Over 100million tonnes of nitrogen (N) fertiliser are applied globally each year to maintain high yields in agricultural crops. The rising price of N fertilisers has made them a major cost for farmers. Inefficient use of N fertiliser leads to substantial environmental problems through contamination of air and water resources and can be a significant economic cost. Consequently, there is considerable need to improve the way N fertiliser is used in farming systems. The efficiency with which crops use applied N fertiliser - the nitrogen-use efficiency (NUE) - is currently quite low for cereals. This is the case in both high yielding environments and lower yielding environments characteristic of cereal growing regions of Australia. Multiple studies have attempted to identify the genetic basis of NUE, but the utility of the results is limited because of the complex nature of the trait and the magnitude of genotype by environment interaction. Transgenic approaches have been applied to improve plant NUE but with limited success, due, in part, to a combination of the complexity of the trait but also due to lack of accurate phenotyping methods. This review documents these two approaches and suggests future directions in improving cereal NUE with a focus on the Australian cereal industry.