The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress

In most plants, constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub‐group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3‐enriched Lactococcus lactis membranes showed that this protein is endowed with transport activity, displaying a strong selectivity for against Cl^?. In response to salt stress, translocation to shoots is reduced, at least partly because expression of the root stele transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss‐of‐function mutation in NPF2.3 resulted in decreased root‐to‐shoot translocation and reduced shoot content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na⁺ in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to translocation to the shoots is quantitatively and physiologically significant under salinity.     

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide‐dependent mechanism

Protein tyrosine (Tyr) nitration is a post‐translational modification yielding 3‐nitrotyrosine (NO₂–Tyr). Formation of NO₂–Tyr is generally considered as a marker of nitro‐oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O₂ transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO₂–Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO₂–Tyr25 and NO₂–Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and by alternative means to nitrate reductase, probably via a ˙NO synthase‐like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires  + H₂O₂ and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to . Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO₂–Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.     

Emerging roles for carbonic anhydrase in mesophyll conductance and photosynthesis

Carbonic anhydrase (CA) is an abundant protein in most photosynthesizing organisms and higher plants. This review paper considers the physiological importance of the more abundant CA isoforms in photosynthesis, through their effects on CO₂ diffusion and other processes in photosynthetic organisms. In plants, CA has multiple isoforms in three different families (α, β and γ) and is mainly known to catalyze the CO₂ equilibrium. This reversible conversion has a clear role in photosynthesis, primarily through sustaining the CO₂ concentration at the site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco). Despite showing the same major reaction mechanism, the three main CA families are evolutionarily distinct. For different CA isoforms, cellular localization and total gene expression as a function of developmental stage are predicted to determine the role of each family in relation to the net assimilation rate. Reaction–diffusion modeling and observational evidence support a role for CA activity in reducing resistance to CO₂ diffusion inside mesophyll cells by facilitating CO₂ transfer in both gas and liquid phases. In addition, physical and/or biochemical interactions between CAs and other membrane‐bound compartments, for example aquaporins, are suggested to trigger a CO₂‐sensing response by stomatal movement. In response to environmental stresses, changes in the expression level of CAs and/or stimulated deactivation of CAs may correspond with lower photosynthetic capacity. We suggest that further studies should focus on the dynamics of the relationship between the activity of CAs (with different subcellular localization, abundance and gene expression) and limitations due to CO₂ diffusivity through the mesophyll and supply of CO₂ to photosynthetic reactions.     

Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana

Availability of plant‐specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measured in vitro, often under non‐physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximal in vivo catalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome‐b6f complex, ATP‐citrate synthase, sucrose‐phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition‐specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements from Arabidopsis thaliana rosette with and fluxes through canonical pathways in a constraint‐based model of leaf metabolism. In comparison to findings in Escherichia coli, we demonstrate weaker concordance between the plant‐specific in vitro and in vivo enzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximal in vivo catalytic rates, and available quantitative metabolomics data are below reported values and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome‐wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.     

High light‐induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability

The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO₂ availability putatively includes enhanced ROS production in the so‐called Mehler reaction. Such conditions are thought to encourage O₂ to become an electron acceptor at photosystem I, producing the ROS superoxide anion radical () and hydrogen peroxide (H₂O₂). In contrast, here it is shown in Chlamydomonas reinhardtii that CO₂ depletion under high light levels lowered cellular H₂O₂ production, and that elevated CO₂ levels increased H₂O₂ production. Using various photosynthetic and mitochondrial mutants of C. reinhardtii, the chloroplast was identified as the main source of elevated H₂O₂ production under high CO₂ availability. High light levels under low CO₂ availability induced photoprotective mechanisms called non‐photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE‐deficient mutant npq4 produced more H₂O₂ than wild‐type cells under high light levels, although less so under high CO₂ availability, whereas it demonstrated equal or greater enzymatic H₂O₂‐degrading capacity. The qT‐deficient mutant stt7‐9 produced the same H₂O₂ as wild‐type cells under high CO₂ availability. Physiological levels of H₂O₂ were able to hinder qT and the induction of state 2, providing an explanation for why under high light levels and high CO₂ availability wild‐type cells behaved like stt7‐9 cells stuck in state 1.     

Soil moisture variations affect short‐term plant‐microbial competition for ammonium,glycine, and glutamate

We tested whether the presence of plant roots would impair the uptake of ammonium (), glycine, and glutamate by microorganisms in a deciduous forest soil exposed to constant or variable moisture in a short‐term (24‐h) experiment. The uptake of ¹⁵NH₄ and dual labeled amino acids by the grass Festuca gigantea L. and soil microorganisms was determined in planted and unplanted soils maintained at 60% WHC (water holding capacity) or subject to drying and rewetting. The experiment used a design by which competition was tested in soils that were primed by plant roots to the same extent in the planted and unplanted treatments. Festuca gigantea had no effect on microbial N uptake in the constant moist soil, but its presence doubled the microbial uptake in the dried and rewetted soil compared with the constant moist. The drying and rewetting reduced by half or more the uptake by F. gigantea, despite more than 60% increase in the soil concentration of . At the same time, the amino acid and ‐ N became equally valued in the plant uptake, suggesting that plants used amino acids to compensate for the lower acquisition. Our results demonstrate the flexibility in plant‐microbial use of different N sources in response to soil moisture fluctuations and emphasize the importance of including transient soil conditions in experiments on resource competition between plants and soil microorganisms. Competition between plants and microorganisms for N is demonstrated by a combination of removal of one of the potential competitors, the plant, and subsequent observations of the uptake of N in the organisms in soils that differ only in the physical presence and absence of the plant during a short assay. Those conditions are necessary to unequivocally test for competition.     

Contemporary effective population and metapopulation size (Ne and meta‐Ne): comparison among three salmonids inhabiting a fragmented system and differing in gene flow and its asymmetries

We estimated local and metapopulation effective sizes ( and meta‐) for three coexisting salmonid species (Salmo salar, Salvelinus fontinalis, Salvelinus alpinus) inhabiting a freshwater system comprising seven interconnected lakes. First, we hypothesized that might be inversely related to within‐species population divergence as reported in an earlier study (i.e., F_ST: S. salar> S. fontinalis> S. alpinus). Using the approximate Bayesian computation method implemented in ONeSAMP, we found significant differences in () between species, consistent with a hierarchy of adult population sizes (). Using another method based on a measure of linkage disequilibrium (LDNE: ), we found more finite values for S. salar than for the other two salmonids, in line with the results above that indicate that S. salar exhibits the lowest among the three species. Considering subpopulations as open to migration (i.e., removing putative immigrants) led to only marginal and non‐significant changes in , suggesting that migration may be at equilibrium between genetically similar sources. Second, we hypothesized that meta‐ might be significantly smaller than the sum of local s (null model) if gene flow is asymmetric, varies among subpopulations, and is driven by common landscape features such as waterfalls. One ‘bottom‐up’ or numerical approach that explicitly incorporates variable and asymmetric migration rates showed this very pattern, while a number of analytical models provided meta‐ estimates that were not significantly different from the null model or from each other. Our study of three species inhabiting a shared environment highlights the importance and utility of differentiating species‐specific and landscape effects, not only on dispersal but also in the demography of wild populations as assessed through local s and meta‐s and their relevance in ecology, evolution and conservation.     

Nitrogen fixation by the diazotroph Cylindrospermopsis raciborskii (Cyanophyceae)

Nitrogen fixation has been proposed as a mechanism that allows the diazotrophic cyanobacterium, Cylindrospermopsis raciborskii, to bloom in nitrogen‐limited freshwater systems. However, it is unclear whether dinitrogen fixation (N₂ fixation) can supplement available dissolved inorganic nitrogen (DIN) for growth, or only provides minimum nitrogen (N) for cell maintenance under DIN deplete conditions. Additionally, the rate at which cells can switch between DIN use and N₂ fixation is unknown. This study investigated N₂ fixation under a range of nitrate concentrations. Cultures were grown with pretreatments of nitrate replete (single dose 941 μmol  · L^?1) and N‐free conditions and then either received a single dose of 941 μmol  · L^?1 (N941), 118 μmol  · L^?1 (N118) or 0 N. Heterocysts appeared from days 3 to 5 when treatments of high were transferred to N free media (N941:N0), and from day 5 in N941 transferred to N118 treatments. Conversely, transferring cells from N0 to N941 resulted in heterocysts being discarded from day 3 and day 5 for N0:N118. Heterocyst appearance correlated with a detectable rate of N₂ fixation and up‐regulation of nifH gene expression, the discard of heterocysts occurred after sequential reduction of nifH expression and N₂ fixation. Nitrate uptake rates were not affected by pretreatment, suggesting no regulation or saturation of this uptake pathway. These data demonstrate that for C. raciborskii, N₂ fixation is regulated by the production or discard of heterocysts. In conclusion, this study has shown that N₂ fixation only provides enough N to support relatively low growth under N‐limited conditions, and does not supplement available nitrate to increase growth rates.     

pOsNAR2.1:OsNAR2.1 expression enhances nitrogen uptake efficiency and grain yield in transgenic rice plants

The nitrate () transporter has been selected as an important gene maker in the process of environmental adoption in rice cultivars. In this work, we transferred another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants. The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level, compared with wild type (WT), and ¹⁵N influx in roots increased 21%–32% in response to 0.2 mm and 2.5 mm and 1.25 mm ¹⁵NH₄¹⁵NO₃. Under these three N conditions, the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143%, 129% and 51%, and total N content increased 161%, 242% and 69%, respectively, compared to WT. Furthermore in field experiments we found the grain yield, agricultural nitrogen use efficiency (ANUE), and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21%, 22% and 21%, compared to WT. We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field, found that postanthesis N uptake differed significantly between them, and in comparison with the WT. Postanthesis N uptake (PANU) increased approximately 39% and 85%, in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines, respectively, possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage. These results show that rice NO₃^– uptake, yield and NUE were improved by increased OsNAR2.1 expression via its native promoter.     

Agronomic nitrogen‐use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter

The importance of the nitrate () transporter for yield and nitrogen‐use efficiency (NUE) in rice was previously demonstrated using map‐based cloning. In this study, we enhanced the expression of the OsNRT2.1 gene, which encodes a high‐affinity transporter, using a ubiquitin (Ubi) promoter and the ‐inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants. Transgenic lines expressing pUbi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited the increased total biomass including yields of approximately 21% and 38% compared with wild‐type (WT) plants. The agricultural NUE (ANUE) of the pUbi:OsNRT2.1 lines decreased to 83% of that of the WT plants, while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128% of that of the WT plants. The dry matter transfer into grain decreased by 68% in the pUbi:OsNRT2.1 lines and increased by 46% in the pOsNAR2.1:OsNRT2.1 lines relative to the WT. The expression of OsNRT2.1 in shoot and grain showed that Ubi enhanced OsNRT2.1 expression by 7.5‐fold averagely and OsNAR2.1 promoters increased by about 80% higher than the WT. Interestingly, we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines; however, for pOsNAR2.1:OsNRT2.1 lines, OsNAR2.1 expression was only increased in root, leaf sheaths and internodes. We show that increased expression of OsNRT2.1, especially driven by OsNAR2.1 promoter, can improve the yield and NUE in rice.     

Taxonomic patterns in the nitrogen assimilation of soil prokaryotes

Nitrogen (N) is frequently a limiting nutrient in soil; its availability can govern ecosystem functions such as primary production and decomposition. Assimilation of N by microorganisms impacts the availability of N in soil. Despite its established ecological significance, the contributions of microbial taxa to N assimilation are unknown. Here we measure N uptake and use by microbial phylotypes and taxonomic groups within a diverse assemblage of soil microbes through quantitative stable isotope probing (qSIP) with ¹⁵N. Following incubation with ¹⁵ , distinct patterns of ¹⁵N assimilation among taxonomic groups were observed. For instance, glucose addition stimulated ¹⁵N assimilation in most members of Actinobacteria and Proteobacteria but generally decreased ¹⁵N use by Firmicutes and Bacteriodetes. While is considered a preferred and universal source of N to prokaryotes, the majority (> 80%) of N assimilation in our soils could be attributed to a handful of active orders. Characterizing N assimilation of taxonomic groups with ¹⁵N qSIP may provide a basis for understanding how microbial community composition influences N availability in the environment.     

Comparative effects of ammonium,nitrate and urea on growth and photosynthetic efficiency of three bloom‐forming cyanobacteria

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Nitrate or ammonium: Influences of nitrogen source on the physiology of a green alga

In freshwaters, algal species are exposed to different inorganic nitrogen (N_i) sources whose incorporation varies in biochemical energy demand. We hypothesized that due to the lesser energy requirement of ammonium ()‐use, in contrast to nitrate ()‐use, more energy remains for other metabolic processes, especially under CO₂‐ and phosphorus (P_i) limiting conditions. Therefore, we tested differences in cell characteristics of the green alga Chlamydomonas acidophila grown on or under covariation of CO₂ and P_i‐supply in order to determine limitations, in a full‐factorial design. As expected, results revealed higher carbon fixation rates for ‐grown cells compared to growth with under low CO₂ conditions. ‐grown cells accumulated more of the nine analyzed amino acids, especially under P_i‐limited conditions, compared to cells provided with . This is probably due to a slower protein synthesis in cells provided with . In contrast to our expectations, compared to ‐grown cells ‐grown cells had higher photosynthetic efficiency under P_i‐limitation. In conclusion, growth on the N_i‐source did not result in a clearly enhanced C_i‐assimilation, as it was highly dependent on P_i and CO₂ conditions (replete or limited). Results are potentially connected to the fact that C. acidophila is able to use only CO₂ as its inorganic carbon (C_i) source.     

Redox regulation of plant stem cell fate

Despite the importance of stem cells in plant and animal development, the common mechanisms of stem cell maintenance in both systems have remained elusive. Recently, the importance of hydrogen peroxide (H₂O₂) signaling in priming stem cell differentiation has been extensively studied in animals. Here, we show that different forms of reactive oxygen species (ROS) have antagonistic roles in plant stem cell regulation, which were established by distinct spatiotemporal patterns of ROS‐metabolizing enzymes. The superoxide anion () is markedly enriched in stem cells to activate WUSCHEL and maintain stemness, whereas H₂O₂ is more abundant in the differentiating peripheral zone to promote stem cell differentiation. Moreover, H₂O₂ negatively regulates biosynthesis in stem cells, and increasing H₂O₂ levels or scavenging leads to the termination of stem cells. Our results provide a mechanistic framework for ROS‐mediated control of plant stem cell fate and demonstrate that the balance between and H₂O₂ is key to stem cell maintenance and differentiation.     

Patterns of molecular evolution in dioecious and non‐dioecious Silene

Dioecy (i.e. having separate sexes) is a rather rare breeding system in flowering plants. Such rareness may result from a high probability of extinction in dioecious species because of less efficient dispersal and the costs of sexual selection, which are expected to harm dioecious species' survival on the long term. These handicaps should decrease the effective population size () of dioecious species, which in turn should reduce the efficacy of selection. Moreover, sexual selection in dioecious species is expected to specifically affect some genes, which will evolve under positive selection. The relative contribution of these effects is currently unknown and we tried to disentangle them by comparing sequence evolution between dioecious and non‐dioecious species in the Silene genus (Caryophyllaceae), where dioecy has evolved at least twice. For the dioecious species in the section Melandrium, where dioecy is the oldest, we found a global reduction of purifying selection, while on some, male‐biased genes, positive selection was found. For section Otites, where dioecy evolved more recently, we found no significant differences between dioecious and non‐dioecious species. Our results are consistent with the view that dioecy is an evolutionary dead end in flowering plants, although other scenarios for explaining reduced cannot be ruled out. Our results also show that contrasting forces act on the genomes of dioecious plants, and suggest that some time is required before the genome of such plants bears the footprints of dioecy.     

Soil warming opens the nitrogen cycle at the alpine treeline

Climate warming may alter ecosystem nitrogen (N) cycling by accelerating N transformations in the soil, and changes may be especially pronounced in cold regions characterized by N‐poor ecosystems. We investigated N dynamics across the plant–soil continuum during 6 years of experimental soil warming (2007–2012; +4 °C) at a Swiss high‐elevation treeline site (Stillberg, Davos; 2180 m a.s.l.) featuring Larix decidua and Pinus uncinata. In the soil, we observed considerable increases in the pool size in the first years of warming (by >50%), but this effect declined over time. In contrast, dissolved organic nitrogen (DON) concentrations in soil solutions from the organic layer increased under warming, especially in later years (maximum of +45% in 2012), suggesting enhanced DON leaching from the main rooting zone. Throughout the experimental period, foliar N concentrations showed species‐specific but small warming effects, whereas δ¹⁵N values showed a sustained increase in warmed plots that was consistent for all species analysed. The estimated total plant N pool size at the end of the study was greater (+17%) in warmed plots with Pinus but not in those containing Larix, with responses driven by trees. Irrespective of plot tree species identity, warming led to an enhanced N pool size of Vaccinium dwarf shrubs, no change in that of Empetrum hermaphroditum (dwarf shrub) and forbs, and a reduction in that of grasses, nonvascular plants, and fine roots. In combination, higher foliar δ¹⁵N values and the transient response in soil inorganic N indicate a persistent increase in plant‐available N and greater cumulative plant N uptake in warmer soils. Overall, greater N availability and increased DON concentrations suggest an opening of the N cycle with global warming, which might contribute to growth stimulation of some plant species while simultaneously leading to greater N losses from treeline ecosystems and possibly other cold biomes.