Anthony J. Miller

Plant Nitrogen Assimilation and Use Efficiency

Crop productivity relies heavily on nitrogen (N) fertilization. Production and application of N fertilizers consume huge amounts of energy, and excess is detrimental to the environment; therefore, increasing plant N use efficiency (NUE) is essential for the development of sustainable agriculture. Plant NUE is inherently complex, as each step-including N uptake, translocation, assimilation, and remobilization-is governed by multiple interacting genetic and environmental factors. The limiting factors in plant metabolism for maximizing NUE are different at high and low N supplies, indicating great potential for improving the NUE of current cultivars, which were bred in well-fertilized soil. Decreasing environmental losses and increasing the productivity of crop-acquired N requires the coordination of carbohydrate and N metabolism to give high yields. Increasing both the grain and N harvest index to drive N acquisition and utilization are important approaches for breeding future high-NUE cultivars.

Root Nitrogen Acquisition and Assimilation

Nitrogen (N) is the main mineral element in plant tissues and almost all of this nutrient is acquired from the soil by the roots. Nitrogen is available in many different forms in the soil, but the three most abundant forms are nitrate, ammonium and amino acids. The relative importance of these different soil N pools to a plant is difficult to measure and depends on many different environmental factors. Changes in the available amounts and imbalance in the supply of some N forms can even be toxic to plants and in extreme cases can lead to changes in the vegetation. However, the importance of this element for agriculture is reflected in the amounts of N-fertiliser applied to crops and this is a major cost (economic and environmental) for world agriculture. This review covers the molecular mechanisms that the plant uses for accessing these soil N pools and briefly includes consideration of the root N assimilatory pathways that exist in the plant. The soil forms of N that are used by plants depend on many factors, but a series of different transporter and assimilatory genes that can provide access to these pools have been identified. This information can now provide the molecular tools to identify the N sources accessed by a plant and the relative importance of these different pools.

Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels.

Calcium can ameliorate Na+ toxicity in plants by decreasing Na+ influx through nonselective cation channels. Here, we show that elevated external [Ca2+] also inhibits Na+-induced K+ efflux through outwardly directed, K+-permeable channels. Noninvasive ion flux measuring and patch-clamp techniques were used to characterize K+ fluxes from Arabidopsis (Arabidopsis thaliana) root mature epidermis and leaf mesophyll under various Ca2+ to Na+ ratios. NaCl-induced K+ efflux was not related to the osmotic component of the salt stress, was inhibited by the K+ channel blocker TEA+, was not mediated by inwardly directed K+ channels (tested in the akt1 mutant), and resulted in a significant decrease in cytosolic K+ content. NaCl-induced K+ efflux was partially inhibited by 1 mm Ca2+ and fully prevented by 10 mm Ca2+. This ameliorative effect was at least partially attributed to a less dramatic NaCl-induced membrane depolarization under high Ca2+ conditions. Patch-clamp experiments (whole-cell mode) have demonstrated that two populations of Ca2+-sensitive K+ efflux channels exist in protoplasts isolated from the mature epidermis of Arabidopsis root and leaf mesophyll cells. The instantaneously activating K+ efflux channels showed weak voltage dependence and insensitivity to external and internal Na+. Another population of K+ efflux channels was slowly activating, steeply rectifying, and highly sensitive to Na+. K+ efflux channels in roots and leaves showed different Ca2+ and Na+ sensitivities, suggesting that these organs may employ different strategies to withstand salinity. Our results suggest an additional mechanism of Ca2+ action on salt toxicity in plants: the amelioration of K+ loss from the cell by regulating (both directly and indirectly) K+ efflux channels.

Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities

This review evaluates the importance of root exudates in determining rhizosphere bacterial community structure. We present evidence that indicates that: (1) the direct influence of root exudates on rhizosphere bacterial communities is limited to small spatiotemporal windows related to root apices; (2) upon rapid assimilation by microorganisms, root exudates are modified, independent of plant influences, before rerelease into the rhizosphere by the microorganisms themselves--thus, at short distances from root apices, rhizosphere carbon pools are unlikely to be dominated by root exudates; and (3) many of the major compounds found in root exudates are ubiquitous in the rhizosphere as they are found in other pools of rhizodeposits and in microbial exudates. Following this argument, we suggest that the importance of root exudates in structuring rhizosphere bacterial communities needs to be considered in the context of the wider contribution of other rhizosphere carbon pools. Finally, we discuss the implications of rhizosphere bacterial distribution trends for the development of effective strategies to manage beneficial plant-microorganism interactions.

Two rice phosphate transporters, OsPht1;2 and OsPht1;6, have different functions and kinetic properties in uptake and translocation.

Plant phosphate (Pi) transporters mediate the uptake and translocation of this nutrient within plants. A total of 13 sequences in the rice (Oryza sativa) genome can be identified as belonging to the Pi transporter (Pht1) family. Here, we report on the expression patterns, biological properties and the physiological roles of two members of the family: OsPht1;2 (OsPT2) and OsPht1;6 (OsPT6). Expression of both genes increased significantly under Pi deprivation in roots and shoots. By using transgenic rice plants expressing the GUS reporter gene, driven by their promoters, we detected that OsPT2 was localized exclusively in the stele of primary and lateral roots, whereas OsPT6 was expressed in both epidermal and cortical cells of the younger primary and lateral roots. OsPT6, but not OsPT2, was able to complement a yeast Pi uptake mutant in the high-affinity concentration range. Xenopus oocytes injected with OsPT2 mRNA showed increased Pi accumulation and a Pi-elicited depolarization of the cell membrane electrical potential, when supplied with mM external concentrations. Both results show that OsPT2 mediated the uptake of Pi in oocytes. In transgenic rice, the knock-down of either OsPT2 or OsPT6 expression by RNA interference significantly decreased both the uptake and the long-distance transport of Pi from roots to shoots. Taken together, these data suggest OsPT6 plays a broad role in Pi uptake and translocation throughout the plant, whereas OsPT2 is a low-affinity Pi transporter, and functions in translocation of the stored Pi in the plant.

Arabidopsis root K+-efflux conductance activated by hydroxyl radicals: single-channel properties, genetic basis and involvement in stress-induced cell death

Reactive oxygen species (ROS) are central to plant stress response, signalling, development and a multitude of other processes. In this study, the plasma-membrane hydroxyl radical (HR)-activated K+ channel responsible for K+ efflux from root cells during stress accompanied by ROS generation is characterised. The channel showed 16-pS unitary conductance and was sensitive to Ca2+, tetraethylammonium, Ba2+, Cs+ and free-radical scavengers. The channel was not found in the gork1-1 mutant, which lacks a major plasma-membrane outwardly rectifying K+ channel. In intact Arabidopsis roots, both HRs and stress induced a dramatic K+ efflux that was much smaller in gork1-1 plants. Tests with electron paramagnetic resonance spectroscopy showed that NaCl can stimulate HR generation in roots and this might lead to K+-channel activation. In animals, activation of K+-efflux channels by HRs can trigger programmed cell death (PCD). PCD symptoms in Arabidopsis roots developed much more slowly in gork1-1 and wild-type plants treated with K+-channel blockers or HR scavengers. Therefore, similar to animal counterparts, plant HR-activated K+ channels are also involved in PCD. Overall, this study provides new insight into the regulation of plant cation transport by ROS and demonstrates possible physiological properties of plant HR-activated K+ channels.

Single-Cell Measurements of the Contributions of Cytosolic Na+ and K+ to Salt Tolerance

Ion concentrations in the roots of two barley (Hordeum vulgare) varieties that differed in NaCl tolerance were compared after exposure to NaCl. Triple-barreled H+-, K+-, and Na+-selective microelectrodes were used to measure cytosolic activities of the three ions after 5 and 8 d of NaCl stress. In both varieties of barley, it was only possible to record successfully from root cortical cells because the epidermal cells appeared to be damaged. The data show that from the 1st d of full NaCl stress, there were differences in the way in which the two varieties responded. At 5 d, the tolerant variety maintained a 10-fold lower cytosolic Na+ than the more sensitive variety, although by 8 d the two varieties were not significantly different. At this time, the more tolerant variety was better at maintaining root cytosolic K+ in the high-NaCl background than was the more sensitive variety. In contrast to earlier work on K+-starved barley (Walker et al., 1996), there was no acidification of the cytosol associated with the decreased cytosolic K+ activity during NaCl stress. These single-cell measurements of cytosolic and vacuolar ion activities allow calculation of thermodynamic gradients that can be used to reveal (or predict) the type of active transporters at both the plasma membrane and tonoplast.

Characterization of a Two-Component High-Affinity Nitrate Uptake System in Arabidopsis. Physiology and Protein-Protein Interaction

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 × atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.

Multilevel Analysis of Primary Metabolism Provides New Insights into the Role of Potassium Nutrition for Glycolysis and Nitrogen Assimilation in Arabidopsis Roots

Potassium (K) is required in large quantities by growing crops, but faced with high fertilizer prices, farmers often neglect K application in favor of nitrogen and phosphorus. As a result, large areas of farmland are now depleted of K. K deficiency affects the metabolite content of crops with negative consequences for nutritional quality, mechanical stability, and pathogen/pest resistance. Known functions of K in solute transport, protein synthesis, and enzyme activation point to a close relationship between K and metabolism, but it is unclear which of these are the most critical ones and should be targeted in biotechnological efforts to improve K usage efficiency. To identify metabolic targets and signaling components of K stress, we adopted a multilevel approach combining transcript profiles with enzyme activities and metabolite profiles of Arabidopsis (Arabidopsis thaliana) plants subjected to low K and K resupply. Roots and shoots were analyzed separately. Our results show that regulation of enzymes at the level of transcripts and proteins is likely to play an important role in plant adaptation to K deficiency by (1) maintaining carbon flux into amino acids and proteins, (2) decreasing negative metabolic charge, and (3) increasing the nitrogen-carbon ratio in amino acids. However, changes in transcripts and enzyme activities do not explain the strong and reversible depletion of pyruvate and accumulation of sugars observed in the roots of low-K plants. We propose that the primary cause of metabolic disorders in low-K plants resides in the direct inhibition of pyruvate kinase activity by low cytoplasmic K in root cells.

Methylated arsenic species in plants originate from soil microorganisms

• Inorganic arsenic (iAs) is a ubiquitous human carcinogen, and rice (Oryza sativa) is the main contributor to iAs in the diet. Methylated pentavalent As species are less toxic and are routinely found in plants; however, it is currently unknown whether plants are able to methylate As. • Rice, tomato (Solanum lycopersicum) and red clover (Trifolium pratense) were exposed to iAs, monomethylarsonic acid (MMA(V)), or dimethylarsinic acid (DMA(V)), under axenic conditions. Rice seedlings were also grown in two soils under nonsterile flooded conditions, and rice plants exposed to arsenite or DMA(V) were grown to maturity in nonsterile hydroponic culture. Arsenic speciation in samples was determined by HPLC-ICP-MS. • Methylated arsenicals were not found in the three plant species exposed to iAs under axenic conditions. Axenically grown rice was able to take up MMA(V) or DMA(V), and reduce MMA(V) to MMA(III) but not convert it to DMA(V). Methylated As was detected in the shoots of soil-grown rice, and in rice grain from nonsterile hydroponic culture. GeoChip analysis of microbial genes in a Bangladeshi paddy soil showed the presence of the microbial As methyltransferase gene arsM. • Our results suggest that plants are unable to methylate iAs, and instead take up methylated As produced by microorganisms.