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Background The soil environment is responsible for sustaining most terrestrial plant life, yet we know surprisingly little about the important functions carried out by diverse microbial communities in soil. Soil microbes that inhabit the channels of decaying root systems, the detritusphere, are likely to be essential for plant growth and health, as these channels are the preferred locations of new root growth. Understanding the microbial metagenome of the detritusphere, and how it responds to agricultural management such as crop rotations and soil tillage, is vital for improving global food production. Results This study establishes an in-depth soil microbial gene catalogue based on the living-decaying rhizosphere niches in a cropping soil. The detritusphere microbiome regulates the composition and function of the rhizosphere microbiome to a greater extent than plant type: rhizosphere microbiomes of wheat and chickpea were homogenous (65–87% similarity) in the presence of decaying root (DR) systems but were heterogeneous (3–24% similarity) where DR was disrupted by tillage. When the microbiomes of the rhizosphere and the detritusphere interact in the presence of DR, there is significant degradation of plant root exudates by the rhizosphere microbiome, and genes associated with membrane transporters, carbohydrate and amino acid metabolism are enriched. Conclusions The study describes the diversity and functional capacity of a high-quality soil microbial metagenome. The results demonstrate the contribution of the detritusphere microbiome in determining the metagenome of developing root systems. Modifications in root microbial function through soil management can ultimately govern plant health, productivity and food security.

The mechanisms controlling the genesis of rhizosheaths are not well understood, despite their importance in controlling the flux of nutrients and water from soil to root. Here, we examine the development of rhizosheaths from drought-tolerant and drought-sensitive chickpea varieties; focusing on the three-dimensional characterization of the pore volume (> 16 mu m voxel spatial resolution) obtained from X-ray microtomography, along with the characterization of mucilage and root hairs, and water sorption. We observe that drought-tolerant plants generate a larger diameter root, and a greater and more porous mass of rhizosheath, which also has a significantly increased water sorptivity, as compared with bulk soil. Using lattice Boltzmann simulations of soil permeability, we find that the root activity of both cultivars creates an anisotropic structure in the rhizosphere, in that its ability to conduct water in the radial direction is significantly higher than in the axial direction, especially in the drought-tolerant cultivar. We suggest that significant differences in rhizosheath architectures are sourced not only by changes in structure of the volumes, but also from root mucilage, and further suggest that breeding for rhizosheath architectures and function may be a potential future avenue for better designing crops in a changing environment.

Nitrate uptake by plant cells requires both high- and low-affinity transport activities. Arabidopsis thaliana nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high- and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize (Zea mays) homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH-dependent nonbiphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased Km, whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.

Increased mesophyll conductance (gm) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions.

The nitrate transporter 1/peptide transporter (NPF) family represents a growing list of putative nitrate permeable transport proteins expressed within multiple cell types and tissues across a diverse range of plant species. Their designation as nitrate permeable and/or selective transporters is slowly being defined as more genes are characterized and their functional activities tested both in planta and in vitro. The most notable of the NPF family has been the Arabidopsis thaliana homolog, AtNPF6.3, previously known as AtNRT1.1 or CHL1. AtNPF6.3 has traditionally been characterized as a dual-affinity nitrate transporter contributing to root nitrate uptake in Arabidopsis. It has also been identified as a nitrate sensor which regulates the expression of high-affinity nitrate transport proteins NRT2s and lateral root development as a part of the primary nitrate response in plants. The sensor function of AtNPF6.3 has also been attributed to its auxin transport activity. Other homologs of AtNPF6.3 are now being described highlighting the variability in their functional capabilities (alternative substrates and kinetics) linking to structural aspects of the proteins. This review focusses on NPF6.3-like transport proteins and the knowledge that has been gained since their initial discovery over two decades ago. The review will investigate from a structural point of view how NPF6.3-like proteins may transport nitrate as well as other ions and what can be learned from structural uniqueness about predicted activities in plants.

In root nodules rhizobia enter host cells via infection threads. The release of bacteria to a host cell is possible from cell wall-free regions of the infection thread. We hypothesized that the VAMP721d and VAMP721e exocytotic pathway, identified before in Medicago truncatula, has a role in the local modification of cell wall during the release of rhizobia. To clarify the role of VAMP721d and VAMP721e we used Glycine max, a plant with a determinate type of nodule. The localization of the main polysaccharide compounds of primary cell walls was analysed in control vs nodules with partially silenced GmVAMP721d. The silencing of GmVAMP721d blocked the release of rhizobia. Instead of rhizobia-containing membrane compartments - symbiosomes - the infected cells contained big clusters of bacteria embedded in a matrix of methyl-esterified and de-methyl-esterified pectin. These clusters were surrounded by a membrane. We found that GmVAMP721d-positive vesicles were not transporting methyl-esterified pectin. We hypothesized that they may deliver the enzymes involved in pectin turnover. Subsequently, we found that GmVAMP721d is partly co-localized with pectate lyase. Therefore, the biological role of VAMP721d may be explained by its action in delivering pectin-modifying enzymes to the site of release.

We report physiological, anatomical and molecular differences in two economically important grapevine (Vitis vinifera L.) cultivars cv. Grenache (near-isohydric) and Chardonnay (anisohydric) in their response to water-stress induced cavitation. The aim of the study was to compare organ vulnerability (petiole and stem) to cavitation by measuring ultrasonic acoustic emissions (UAE) and percent loss of conductance of potted grapevines subject to the onset of water-stress. Leaf (ψL) and stem water potential (ψS), stomatal conductance (gs), transpiration (E), petiole hydraulics (KPet), and xylem diameter were also measured. Chardonnay displayed hydraulic segmentation based on UAE, with cavitation occurring at a less negative ψL in the petiole than in the stem. Vulnerability segmentation was not observed in Grenache, with both petioles and stems equally vulnerable to cavitation. Leaf water potential that induced 50% of maximum UAE was significantly different between petioles and stems in Chardonnay (ψ50Petiole = -1.14 and ψ50Stem = -2.24 MPa) but not in Grenache (ψ50Petiole = -0.73 and ψ50Stem = -0.78 MPa). Grenache stems appeared more susceptible to water-stress induced cavitation than Chardonnay stems. Grenache displayed (on average) a higher KPet likely due to the presence of larger xylem vessels. A close relationship between petiole hydraulic properties and vine water status was observed in Chardonnay but not in Grenache. Transcriptional analysis of aquaporins in the petioles and leaves (VvPIP1;1, VvPIP2;1, VvPIP2;2 VvPIP2;3, VvTIP1;1, and VvTIP2;1) showed differential regulation diurnally and in response to water-stress. VvPIP2;1 showed strong diurnal regulation in the petioles and leaves of both cultivars with expression highest predawn. Expression of VvPIP2;1 and VvPIP2;2 responded to ψL and ψS in both cultivars indicating the expression of these two genes are closely linked to vine water status. Expression of several aquaporin genes correlated with gas exchange measurements, however, these genes differed between cultivars. In summary, the data shows two contrasting responses in petiole hydraulics and aquaporin expression between the near-isohydric cultivar, Grenache and anisohydric cultivar, Chardonnay.

Nitrogen availability and ontogeny both affect the relative growth rate (RGR) of plants. In this study of barley (Hordeum vulgare L.) we determined which growth parameters are affected by nitrate (N) availability, and whether these were confounded by differences in plant size, reflecting differences in growth. Plants were hydroponically grown on six different nitrate (N) concentrations for 28 days, and nine harvests were performed to assess the effect of N on growth parameters. Most growth parameters showed similar patterns of responses to N supply whether compared at common time points or common plant sizes. N had a significant effect on the biomass allocation: increasing N increased leaf mass ratio (LMR) and decreased root mass ratio (RMR). Specific leaf area (SLA) was not significantly affected by N. RGR increased with increasing N supply up to 1 mM, associated with increases in both LMR and net assimilation rate (NAR). Increases in N supply above 1 mM did not increase RGR as increases in LMR were offset by decreases in NAR. The high RGR at suboptimal N supply suggest a higher nitrogen use efficiency (biomass/N supply). The reasons for the homeostasis of growth under suboptimal N levels are discussed.

In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria.

Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.

Grapevine is moderately sensitive to salinity and accumulation of toxic levels of Cl− in leaves is the major reason for salt-induced symptoms. In this study, apoplastic Cl− uptake and transport mechanism(s) were investigated in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance; 1103 Paulsen (salt tolerant) and K 51–40 (salt sensitive). Increased external salinity caused high Cl− accumulation in shoots of the salt sensitive K 51–40 in comparison to Paulsen. Measurement of 15NO3 − net fluxes under high salinity showed that by increasing external Cl− concentrations K 51–40 roots showed reduced NO3 − accumulation. This was associated with increased accumulation of Cl−. In comparison to Paulsen, K 51–40 showed reduced NO3 −/Cl− root selectivity with increased salinity, but Paulsen had lower selectivity over the whole salinity range (0–45 mM). To examine if root hydraulic and permeability characterisations accounted for differences between varieties, the root pressure probe was used on excised roots. This showed that the osmotic Lpr was significantly smaller than hydrostatic Lpr, but no obvious difference was observed between the rootstocks. The reflection coefficient (σ) values (0.48–0.59) were the same for both rootstocks, and root anatomical studies showed no obvious difference in apoplastic barriers of the main and lateral roots. Comparing the uptake of Cl− with an apoplastic tracer, PTS (3-hydroxy-5,8,10-pyrentrisulphonic acid), showed that there was no correlation between Cl− and PTS transport. These results indicated that bypass flow of salts to the xylem is the same for both rootstocks (0.77 ± 0.2 and 1.05 ± 0.12 %) and hence pointed to differences in membrane transport to explain difference in Cl− transport to the shoot.

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13–20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.

We investigated how root hydraulic conductance (normalized to root dry weight, Lo ) is regulated by the shoot. Shoot topping (about 30% reduction in leaf area) reduced Lo of grapevine (Vitis vinifera L.), soybean (Glycine max L.) and maize (Zea mays L.) by 50 to 60%. More detailed investigations with soybean and grapevine showed that the reduction in Lo was not correlated with the reduction in leaf area, and shading or cutting single leaves had a similar effect. Percentage reduction in Lo was largest when initial Lo was high in soybean. Inhibition of Lo by weak acid (low pH) was smaller after shoot damage or leaf shading. The half time of reduction in Lo was approximately 5 min after total shoot decapitation. These characteristics indicate involvement of aquaporins. We excluded phloem-borne signals and auxin-mediated signals. Xylem-mediated hydraulic signals are possible since turgor rapidly decreased within root cortex cells after shoot topping. There was a significant reduction in the expression of several aquaporins in the plasma membrane intrinsic protein (PIP) family of both grapevine and soybean. In soybean, there was a five- to 10-fold reduction in GmPIP1;6 expression over 0.5-1 h which was sustained over the period of reduced Lo .

Root Cl− transport was investigated using 36Cl− flux analysis in two grapevine (Vitis sp.) rootstock hybrids differing in salt tolerance; 1103 Paulsen (salt-tolerant) and K 51–40 (salt sensitive). Initial 36Cl− influx to the root was greater in Paulsen than K 51–40. This flux, attributed to the Cl− influx to the cytoplasm (Φ oc) increased with increasing external concentrations of Cl− for plants adapted to growth in 30 mM NaCl. The concentration kinetics in this high concentration range could be fit to a Michaeils–Menton equation. There was no significant difference between genotypes in Km (28.68 ± 15.76 and 24.27 ± 18.51 mM for Paulsen and K 51–40, respectively), but Paulsen had greater V max (0.127 ± 0.042) compared to K 51–40 (0.059 ± 0.026 μm g−1 FW min−1). In Paulsen, the main root had greater contribution to 36Cl− uptake than lateral roots, there being no significant difference in lateral root influx between the genotypes. 36Cl− transport to the shoot of K 51–40 was greater than for Paulsen. It was estimated that efflux rate from the xylem parenchyma cells to the xylem vessels (Φ cx) in K 51–40 was twice that of Paulsen. Compartmental analysis from 36Cl− efflux kinetics confirmed the larger Φ oc and the higher ratio of main to lateral root Φ oc for Paulsen. Efflux from the cytoplasm (Φ co) was higher than 95 % of Φ oc indicating a high degree of cycling across the plasma membrane in roots at these high external Cl− concentrations. Paulsen appears to keep the cytoplasmic Cl− concentration in roots lower than K 51–40 via greater efflux to the vacuole and to the outside medium. The difference in salt tolerance between the genotypes can be attributed to different Cl− transport properties at the plasma membrane and tonoplast and particularly in Cl− efflux to the xylem.

Nitrogen (N) uptake in most plants is positively correlated to water flow in and through roots. This allows transpiration to drive convection of mobile N sources in the soil to the root surface. Generally N starvation suppresses root hydraulic conductivity (Lpr) while resupply stimulates. However, ammonium and nitrate can give different responses depending on the species, and this may be associated with the form of N that different species prefer to transport and assimilate. Responses in Lpr in the short to medium term are largely explained by aquaporin regulation at both the transcript and post-translational level. Local and systemic signalling is indicated in the regulation of aquaporins. A direct role for NO3 sensing has been shown as well as a role of a high-affinity NO3− transporter (NRT2.1), but the mode of action in post-translational modification is not known. Transcripts of the aquaporins are also altered by N treatments, yet the regulations of these transcripts and the function of the proteins in N transport remain unclear. Further research is required to uncover the signalling and regulation of aquaporins in relation to N transport since modifications to this process may improve N use efficiency.

The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner ‘nutritious seeds for a sustainable future’. A second green revolution is required to ensure food and nutritional security in the face of global climate change. Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming. In addition, a legume-rich diet has health benefits for humans and livestock alike. However, grain legumes form only a minor part of most current human diets and legume crops are greatly under-utilized. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. The current lack of coordinated focus on grain legumes has compromised human health, nutritional security and sustainable food production.

• Interactions between the Arabidopsis NitRate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity nitrate transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for nitrate transport. • Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and nitrate uptake in Xenopus oocytes. • All NRT2s, except for AtNRT2.7, restored growth and β-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 transporters interact strongly with AtNAR2.1. • Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.

Pollen tube observation was used to assess the self- and cross-incompatibility of the olive (Olea europaea L.) cultivars ‘Frantoio’, ‘Koroneiki’ and ‘Kalamata’. The pistils were harvested 1 week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol and observed under a fluorescence microscope. All the cultivars studied were self-incompatible. ‘Frantoio’ (as a host) was incompatible with ‘Koroneiki’ and ‘Barnea’ but partially compatible with ‘Mission’. ‘Koroneiki’ (as a host) was incompatible with ‘Barnea’ but partially compatible with ‘Frantoio’ and ‘Mission’. ‘Kalamata’ (as a host) was compatible with ‘Barnea’, incompatible with ‘Mission’ and ‘Koroneiki’ in 2004, but partially compatible with them in 2005. The effects of shoot orientation and inflorescence location on inflorescence characteristics were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle and base) and four sides ...

Magnesium accumulates at high concentrations in dicotyledonous leaves but it is not known in which leaf cell types it accumulates, by what mechanism this occurs and the role it plays when stored in the vacuoles of these cell types. Cell-specific vacuolar elemental profiles from Arabidopsis thaliana (Arabidopsis) leaves were analysed by X-ray microanalysis under standard and serpentine hydroponic growth conditions and correlated with the cell-specific complement of magnesium transporters identified through microarray analysis and quantitative polymerase chain reaction (qPCR). Mesophyll cells accumulate the highest vacuolar concentration of magnesium in Arabidopsis leaves and are enriched for members of the MGT/MRS2 family of magnesium transporters. Specifically, AtMGT2/AtMRS2-1 and AtMGT3/AtMRS2-5 were shown to be targeted to the tonoplast and corresponding T-DNA insertion lines had perturbed mesophyll-specific vacuolar magnesium accumulation under serpentine conditions. Furthermore, transcript abundance of these genes was correlated with the accumulation of magnesium under serpentine conditions, in a low calcium-accumulating mutant and across 23 Arabidopsis ecotypes varying in their leaf magnesium concentrations. We implicate magnesium as a key osmoticum required to maintain growth in low calcium concentrations in Arabidopsis. Furthermore, two tonoplast-targeted members of the MGT/MRS2 family are shown to contribute to this mechanism under serpentine conditions. Refereed/Peer-reviewed

The influence of flower position on the inflorescence on opening day, gender, and petal persistence was studied in three olive cultivars: Manzanillo, Mission, and Frantoio. In each cultivar, 45 inflorescences were checked every morning from flower opening to petal fall. Perfect flowers opened mainly in the beginning of the flower opening period, and staminate flowers opened later. Flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than the flowers located on the secondary branches. Flower position had also a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. Among the primary branches, the branch arising immediately next to the terminal flower had the latest flowers to open and the lowest percent of perfect flowers. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal half of 120 inflorescences, on which the flowers tend to be perfect, in three trees of Manzanillo were removed about 1 month before full bloom. There was a highly significant effect on the percent of perfect flowers that opened on the proximal half. Flower competition may be a reason for pistil abortion in flowers located on secondary branches.

Refereed/Peer-reviewed Elucidation of the gene networks underlying the response to N supply and demand will facilitate the improvement of the N uptake efficiency of plants. We undertook a transcriptomic analysis of maize to identify genes responding to both a non-growth-limiting decrease in NO3- provision and to development-based N demand changes at seven representative points across the life cycle. Gene co-expression networks were derived by cluster analysis of the transcript profiles. The majority of NO3--responsive transcription occurred at 11 (D11), 18 (D18) and 29 (D29) days after emergence, with differential expression predominating in the root at D11 and D29 and in the leaf at D18. A cluster of 98 probe sets was identified, the expression pattern of which is similar to that of the high-affinity NO3- transporter (NRT2) genes across the life cycle. The cluster is enriched with genes encoding enzymes and proteins of lipid metabolism and transport, respectively. These are candidate genes for the response of maize to N supply and demand. Only a few patterns of differential gene expression were observed over the entire life cycle; however, the composition of the classes of the genes differentially regulated at individual time points was unique, suggesting tightly controlled regulation of NO3--responsive gene expression. usc

A protocol for Agrobacterium-mediated transformation with either kanamycin or mannose selection was developed for leaf explants of the cultivar Prunus dulcis cv. Ne Plus Ultra. Regenerating shoots were selected on medium containing 15 muM kanamycin (negative selection), while in the positive selection strategy, shoots were selected on 2.5 g/l mannose supplemented with 15 g/l sucrose. Transformation efficiencies based on PCR analysis of individual putative transformed shoots from independent lines relative to the initial numbers of leaf explants tested were 5.6% for kanamycin/nptII and 6.8% for mannose/pmi selection, respectively. Southern blot analysis on six randomly chosen PCR-positive shoots confirmed the presence of the nptII transgene in each, and five randomly chosen lines identified to contain the pmi transgene by PCR showed positive hybridisation to a pmi DNA probe. The positive (mannose/pmi) and the negative (kanamycin) selection protocols used in this study have greatly improved transformation efficiency in almond, which were confirmed with PCR and Southern blot. This study also demonstrates that in almond the mannose/pmi selection protocol is appropriate and can result in higher transformation efficiencies over that of kanamycin/nptII selection protocols.

• Background The importance of molybdenum for plant growth is disproportionate with respect to the absolute amounts required by most plants. Apart from Cu, Mo is the least abundant essential micronutrient found in most plant tissues and is often set as the base from which all other nutrients are compared and measured. Molybdenum is utilized by selected enzymes to carry out redox reactions. Enzymes that require molybdenum for activity include nitrate reductase, xanthine dehydrogenase, aldehyde oxidase and sulfite oxidase. • Scope Loss of Mo-dependent enzyme activity (directly or indirectly through low internal molybdenum levels) impacts upon plant development, in particular, those processes involving nitrogen metabolism and the synthesis of the phytohormones abscisic acid and indole-3 butyric acid. Currently, there is little information on how plants access molybdate from the soil solution and redistribute it within the plant. In this review, the role of molybdenum in plants is discussed, focusing on its current constraints in some agricultural situations and where increased molybdenum nutrition may aid in agricultural plant development and yields. • Conclusions Molybdenum deficiencies are considered rare in most agricultural cropping areas; however, the phenotype is often misdiagnosed and attributed to other downstream effects associated with its role in various enzymatic redox reactions. Molybdenum fertilization through foliar sprays can effectively supplement internal molybdenum deficiencies and rescue the activity of molybdoenzymes. The current understanding on how plants access molybdate from the soil solution or later redistribute it once in the plant is still unclear; however, plants have similar physiological molybdenum transport phenotypes to those found in prokaryotic systems. Thus, careful analysis of existing prokaryotic molybdate transport mechanisms, as well as a re-examination of know anion transport mechanisms present in plants, will help to resolve how this important trace element is accumulated.

An understanding of the adaptations made by plants in their nitrogen (N) uptake systems in response to reduced N supply is important to the development of cereals with enhanced N uptake efficiency (NUpE). Twenty seven diverse genotypes of maize (Zea mays, L.) were grown in hydroponics for 3 weeks with limiting or adequate N supply. Genotypic response to N was assessed on the basis of biomass characteristics and the activities of the nitrate ([Formula: see text]) and ammonium ([Formula: see text]) high-affinity transport systems. Genotypes differed greatly for the ability to maintain biomass with reduced N. Although, the N response in underlying biomass and N transport related characteristics was less than that for biomass, there were clear relationships, most importantly, lines that maintained biomass at reduced N maintained net N uptake with no change in size of the root relative to the shoot. The root uptake capacity for both [Formula: see text] and [Formula: see text] increased with reduced N. Transcript levels of putative [Formula: see text] and [Formula: see text] transporter genes in the root tissue of a subset of the genotypes revealed that predominately ZmNRT2 transcript levels responded to N treatments. The correlation between the ratio of transcripts of ZmNRT2.2 between the two N levels and a genotype's ability to maintain biomass with reduced N suggests a role for these transporters in enhancing NUpE. The observed variation in the ability to capture N at low N provides scope for both improving NUpE in maize and also to better understand the N uptake system in cereals.

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 13 NH4 influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity 13 NH4 influx in the mutant by 30% compared with wild-type plants, whereas lowaffinity 13 NH4 influx (250 m–10 mm NH4 ) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants 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.

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. Stickstofftransport in Pflanzen unter besonderer Berucksichtigung der Regulation der am N-Bedarf orientierten Fluxe Physiologische Methoden, insbesondere der Einsatz von N-Isotopen, haben eine detaillierte Charakterisierung der verschiedenen mutmaslichen Transportsysteme fur Nitrat und Ammonium in Pflanzenwurzeln ermoglicht. Ein besonderer Fortschritt im letzten Jahrzehnt war die Klonierung von Genen, die hoch- und wenig-affine Transportproteine codieren. Hierdurch konnten Schlussfolgerungen aus fruheren physiologischen Arbeiten untermauert werden. Trotz dieses Erkenntnisfortschritts stellt die un-erwartete genetische Vielfalt die Forschung vor noch grosere Herausforderungen bei der Klarung der einzelnen Funktionen und bei dem Bemuhen, diese im Gesamtkontext Pflanze/Umwelt zu deuten.

Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a novel family of 2OG-sensing proteins – the nitrogen regulatory protein NrpR – that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR re-positions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.

The impact of nitrate (5–15 mM, 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent (Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 mM nitrate, but not 5 mM nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 mM nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 mM) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O2 limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.

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. Refereed/Peer-reviewed

Paternity analysis was used to assess the self-incompatibility of the olive (Olea europaea L.) cultivar ‘Kalamata’ and to identify some compatible pollenisers under a Mediterranean-type climate. Eight microsatellite markers were used for genotyping three ‘Kalamata’ mother trees, 120 embryos, and all potential pollen donors. The identified alleles were analysed using FaMoz software and showed that ‘Kalamata’ was highly self-incompatible. Only three ‘Kalamata’ embryos were assigned to ‘Kalamata’ self-fertilisation, even though it was the most available pollen donor. The alleles were also analysed using NTSYS-pc (version 2.02 k) software and identified 54 potential pollen donors in the study site; however, not all of them were located within the effective pollination distance of the mother trees (30 m in olive). According to the results of this study, ‘Kalamata’ (as a host) was compatible with ‘Barnea’, ‘Benito’, and ‘Katsourela’ (six ‘Kalamata’ embryos assigned in each) but incompatible with ‘Arbequina’, ‘Azapa’, and ‘Picual’ (zero ‘Kalamata’ embryos assigned in each). The olive growers could use some of these compatible pollenisers with ‘Kalamata’ to guarantee good fruit set. Se utilizó un análisis de paternidad para evaluar la auto-incompatibilidad del olivo (Olea europaea L.) 'Kalamata' e identificar, en un clima de tipo mediterráneo, algunos polinizadores compatibles. Se utilizaron ocho marcadores microsatélites para el genotipado de tres árboles madre 'Kalamata', 120 embriones y todos los potenciales donantes de polen. Los alelos identificados fueron analizados mediante el software de FaMoz, que mostró que 'Kalamata' fue altamente auto-incompatible. Sólo tres embriones de 'Kalamata' fueron asignados a la autofecundación de 'Kalamata', a pesar de que fue el donante de polen más disponible. Los alelos también fueron analizados mediante NTSYS-pc, que identificó 54 posibles donantes de polen en el sitio de estudio; sin embargo, no todos ellos se encontraban dentro de la distancia de polinización efectiva de los árboles madre (30 m en olivo). De acuerdo con los resultados de este estudio, 'Kalamata' (como huésped) fue compatible con 'Barnea', 'Benito', y 'Katsourela (seis embriones 'Kalamata' asignados a cada uno), pero incompatible con 'Arbequina', 'Azapa' y 'Picual' (cero embriones 'Kalamata' asignados a cada uno). Los olivareros podrían utilizar algunos de estos polinizadores compatibles con 'Kalamata' para garantizar una buena fructificación.

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis 60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca2+ transporters, CAX1 (Ca2+/H+-antiporter), ACA4, and ACA11 (Ca2+-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO2 assimilation, and leaf growth rate; increased transcript abundance of other Ca2+ transporter genes; altered expression of cell wall–modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca2+], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca2+] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

A large proportion of the nitrate (NO(3)(-)) acquired by plants from soil is actively transported via members of the NRT families of NO(3)(-) 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 NO(3)(-) transporters and NO(3)(-) transport in grass crop species.

Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca 2+ signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca 2+ -dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa. Structured summary MINT- 8051564 : AtCRLK2 (uniprotkb: Q9LFV3 ) binds (MI: 0407 ) to CaM (uniprotkb: P62199 ) by filter binding (MI: 0049 ) MINT- 8051416 : GmCaMK3 (uniprotkb: C6ZRS6 ) binds (MI: 0407 ) to CaM (uniprotkb: P62199 ) by filter binding (MI: 0049 ) MINT- 8051258 : CaM (uniprotkb: P62199 ) and GmCaMK1 (genbank_protein_gi: 223452504 ) bind (MI: 0407 ) by isothermal titration calorimetry (MI: 0065 ) MINT- 8051400 : GmCaMK2 (uniprotkb: C6ZRY5 ) binds (MI: 0407 ) to CaM (uniprotkb: P62199 ) by filter binding (MI: 0049 ) MINT- 8051242 , MINT- 8051295 , MINT- 8051313 , MINT- 8051327 , MINT- 8051341 , MINT- 8051355 : GmCaMK1 (genbank_protein_gi: 223452504 ) binds (MI: 0407 ) to CaM (uniprotkb: P62199 ) by filter binding (MI: 0049 ) MINT- 8051467 : GmCaMK4 (uniprotkb: C6TIQ0 ) binds (MI: 0407 ) to CaM (uniprotkb: P62199 ) by filter binding (MI: 0049 ) MINT- 8051276 : CaM (uniprotkb: P62199 ) and GmCaMK1 (genbank_protein_gi: 223452504 ) bind (MI: 0407 ) by comigration in non denaturing gel electrophoresis (MI: 0404 ) MINT- 8051374 : CaM (uniprotkb: P62199 ) and GmCaMK1 (genbank_protein_gi: 223452504 ) bind (MI: 0407 ) by mass spectrometry studies of complexes (MI: 0069 )

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. Refereed/Peer-reviewed

We report physiological and anatomical characteristics of water transport across roots grown in soil of two cultivars of grapevine (Vitis vinifera) differing in response to water stress (Grenache, isohydric; Chardonnay, anisohydric). Both cultivars have similar root hydraulic conductances (L o; normalized to root dry weight) that change diurnally. There is a positive correlation between L o and transpiration. Under water stress, both cultivars have reduced minimum daily L o (predawn) attributed to the development of apoplastic barriers. Water-stressed and well-watered Chardonnay had the same diurnal change in amplitude of L o, while water-stressed Grenache showed a reduction in daily amplitude compared with well-watered plants. Hydraulic conductivity of root cortex cells (L pcell) doubles in Chardonnay but remains unchanged in Grenache. Of the two most highly expressed plasma membrane intrinsic protein (PIP) aquaporins in roots (VvPIP1;1 and VvPIP2;2), only VvPIP2;2 functions as a water channel in Xenopus laevis oocytes. VvPIP1;1 interacts with VvPIP2;2 to induce 3-fold higher water permeability. These two aquaporins are colocated in the root from in situ hybridization and immunolocalization of VvPIP1 and VvPIP2 subfamily members. They occur in root tip, exodermis, root cortex (detected up to 30 mm), and stele. VvPIP2;2 mRNA does not change diurnally or with water stress, in contrast to VvPIP1;1, in which expression reflects the differences in L o and L pcell between cultivars in their responses to water stress and rewatering. VvPIP1;1 may regulate water transport across roots such that transpirational demand is matched by root water transport capacity. This occurs on a diurnal basis and in response to water stress that corresponds to the difference in drought tolerance between the cultivars.

The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.

Two O2 ramping techniques (linear versus exponential) were used to investigate the response of H2 evolution from intact nodules of soybean (Glycine max (L.) Merr. 'Maple Arrow'), stem-girdled soybean, pea (Pisum sativum L. 'Juneau'), and common bean (Phaseolus vulgaris L. 'Ex Rico 23') to increasing O2 concentrations from 20 to 100% over a 30-min period. The data indicate symbiosis-specific responses to the two ramps, and possible implications for determination of O2 limitation of N2 fixation. Key words: Hydrogen evolution, legume, nitrogen fixation

Inorganic nitrogen concentrations in soil solutions vary across several orders of magnitude among different soils and as a result of seasonal changes. In order to respond to this heterogeneity, plants have evolved mechanisms to regulate and influx. In addition, efflux analysis using (13)N has revealed that there is a co-ordinated regulation of all component fluxes within the root, including biochemical fluxes. Physiological studies have demonstrated the presence of two high-affinity transporter systems (HATS) for and one HATS for in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for and five members of the AMT1 family encoding putative HATS for. The induction of high-affinity transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of, while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated and may act post-transcriptionally on transporter function. The present challenge is to resolve the functions of all of these genes. In Aspergillus nidulans and Chlamydomonas reinhardtii there are but two high-affinity transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of absorption over the wide range of concentration normally found in nature. Such kinetic differentiation may also have occurred among higher plant transporters. The characterization of transporter function in higher plants is currently being inferred from patterns of gene expression in roots and shoots, as well as through studies of heterologous expression systems and knockout mutants.