Development of pENTR-NeCo-lacZα vectors for the preparation of negative control constructs in Gateway cloning.

Gateway cloning is a useful technology for the simple and reliable preparation of various construct in many organisms. However, there is a problem regarding the negative control construct in the Gateway cloning system. In this study, we developed the pENTR-NeCo-lacZα vector system to create an empty vector that can be used as a negative control construct in Gateway cloning.

Expression analysis of genes encoding extracellular leucine-rich repeat proteins in Arabidopsis thaliana.

Leucine-rich repeat (LRR)-containing proteins have been identified in diverse species, including plants. The diverse intracellular and extracellular LRR variants are responsible for numerous biological processes. We analyzed the expression patterns of Arabidopsis thaliana extracellular LRR (AtExLRR) genes, 10 receptor-like proteins, and 4 additional genes expressing the LRR-containing protein by a promoter: ß-glucuronidase (GUS) study. According to in silico expression studies, several AtExLRR genes were expressed in a tissue- or stage-specific and abiotic/hormone stress-responsive manner, indicating their potential participation in specific biological processes. Based on the promoter: GUS assay, AtExLRRs were expressed in different cells and organs. A quantitative real-time PCR investigation revealed that the expressions of AtExLRR3 and AtExLRR9 were distinct under various abiotic stress conditions. This study investigated the potential roles of extracellular LRR proteins in plant growth, development, and response to various abiotic stresses.

Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, affects cytokinesis of guard mother cells and exine formation of pollen in Arabidopsis thaliana.

MAIN CONCLUSION: Targeted expression of bgl23-D, a dominant-negative allele of ATCSLD5, is a useful genetic approach for functional analysis of ATCSLDs in specific cells and tissues in plants. Stomata are key cellular structures for gas and water exchange in plants and their development is influenced by several genes. We found the A. thaliana bagel23-D (bgl23-D) mutant showing abnormal bagel-shaped single guard cells. The bgl23-D was a novel dominant mutation in the A. thaliana cellulose synthase-like D5 (ATCSLD5) gene that was reported to function in the division of guard mother cells. The dominant character of bgl23-D was used to inhibit ATCSLD5 function in specific cells and tissues. Transgenic A. thaliana expressing bgl23-D cDNA with the promoter of stomata lineage genes, SDD1, MUTE, and FAMA, showed bagel-shaped stomata as observed in the bgl23-D mutant. Especially, the FAMA promoter exhibited a higher frequency of bagel-shaped stomata with severe cytokinesis defects. Expression of bgl23-D cDNA in the tapetum with SP11 promoter or in the anther with ATSP146 promoter induced defects in exine pattern and pollen shape, novel phenotypes that were not shown in the bgl23-D mutant. These results indicated that bgl23-D inhibited unknown ATCSLD(s) that exert the function of exine formation in the tapetum. Furthermore, transgenic A. thaliana expressing bgl23-D cDNA with SDD1, MUTE, and FAMA promoters showed enhanced rosette diameter and increased leaf growth. Taken together, these findings suggest that the bgl23-D mutation could be a helpful genetic tool for functional analysis of ATCSLDs and manipulating plant growth.

Root-specific activation of plasma membrane H+-ATPase 1 enhances plant growth and shoot accumulation of nutrient elements under nutrient-poor conditions in Arabidopsis thaliana.

Plasma membrane (PM) H+-ATPase contributes to nutrient uptake and stomatal opening by creating proton gradient across the membrane. Previous studies report that a dominant mutation in the OPEN STOMATA2 locus (OST2-2D) constitutively activates Arabidopsis PM H+-ATPase 1 (AHA1), which enlarges proton motive force for root nutrient uptake. However, the stomatal opening is also constitutively enhanced in the ost2-2D, causing drought hypersensitivity. To develop plants with improved nutrient acquisition and normal stomatal movement, we generated grafted plants (scion/rootstock: Col-0 (WT)/ost2-2D), and compared their growth, nutrient element content, and transcriptomes with those of control plants (WT/WT) under nutrient-rich or nutrient-poor conditions. WT/ost2-2D shoots had larger weights, rosette diameter, leaf blade area, and content of C, N, K, Ca, S, P, Mg, Na, Mn, B, Co, and Mo under nutrient-poor conditions compared with WT/WT shoots. The root weights and primary root length were greater in WT/ost2-2D plants than in WT/WT plants under both nutrient conditions. Root expression of the high-affinity nitrate transporter NRT2.1, potassium transporter HAK5, and divalent cation transporter IRT1 was higher in WT/ost2-2D plants than in WT/WT plants under nutrient-poor conditions. These results suggest that root-specific activation of PM H+-ATPase enhances plant growth by increasing root uptake of nutrient elements under nutrient-poor conditions. Our study presents a novel approach to improving nutrient uptake efficiency in crops for low-input sustainable agriculture.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Expression analysis of plant intracellular Ras-group related leucine-rich repeat proteins (PIRLs) in Arabidopsis thaliana.

Arabidopsis thaliana contains a family of nine genes known as plant intracellular Ras-group related leucine-rich repeat (LRR) proteins (PIRLs). These are structurally similar to animals and fungal LRR proteins and play important roles in developmental pathways. However, to date, no detailed tissue-specific expression analysis of these PIRLs has been performed. Therefore, in this study, we generated promoter:GUS transgenic plants for the nine A. thaliana PIRL genes and identified their expression patterns in seedlings and floral organs at different developmental stages. Most PIRL members showed expression in the root apical region and in the vascular tissue of primary and lateral roots. Shoot apex-specific expression was recorded for PIRL1 and PIRL8. Furthermore, PIRL1, PIRL3, PIRL5, PIRL6, and PIRL7 showed distinct expression patterns in flowers, especially in pollen and anthers. In addition, co-expression network analysis identified cases where PIRLs were co-expressed with other genes known to have specific functions related to growth and development. Taken together, the tissue-specific expression patterns of PIRL genes improve our understanding of the functions of this gene family in plant growth and development.

Root-specific Reduction of Cytokinin Perception Enhances Shoot Growth in Arabidopsis thaliana.

Previous studies suggest that root-derived cytokinins (CKs) contribute to shoot growth via long-distance transport; therefore, we hypothesized that an increase in root-derived CKs enhances shoot growth. To verify this, we grafted Arabidopsis Col-0 (wild type, WT) scion onto rootstock originated from WT or a double-knockout mutant of CK receptors Arabidopsis histidine kinase 2 (AHK2) and AHK3 (ahk2-5 ahk3-7; ahk23) because this mutant overaccumulates CKs in the body probably due to feedback homeostasis regulation. The grafted plants (scion/rootstock: WT/WT and WT/ahk23) were grown in vermiculite pots or solid media for vegetative growth and biochemical analysis. The root-specific deficiency of AHK2 and AHK3 increased root concentrations of trans-zeatin (tZ)-type and N6-(Δ2-isopentenyl) adenine (iP)-type CKs, induced CK biosynthesis genes and repressed CK degradation genes in the root. The WT/ahk23 plants had significantly larger shoot weight, rosette diameter and leaves area than did the WT/WT plants. Shoot concentrations of tZ-type CKs showed increasing trends in the WT/ahk23 plants. Moreover, the root-specific deficiency of AHK2 and AHK3 enhanced shoot growth in the WT scion more strongly than in the ahk23 scion, suggesting that shoot growth enhancement could occur through increased shoot perception of CKs. In the WT/ahk23 shoots compared with the WT/WT shoots, however, induction of most of CK-inducible response regulator genes was not statistically significant. Thus we suggest that the root-specific reduction of CK perception enhances shoot growth only partly by increasing the amount of root-derived tZ-type CKs and their perception by shoots. The unknown mechanism(s) distinct from CK signaling would also be involved in the shoot growth enhancement.

Regulation of ammonium acquisition and use in Oryza longistaminata ramets under nitrogen source heterogeneity.

Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and ammonium-deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the nitrogen-deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin (tZ), a cytokinin, was upregulated in response to the nitrogen supply, but tZ appeared not to target the compensatory regulation. Our results also implied that the O. longistaminata putative ortholog of rice (Oryza sativa) C-terminally encoded peptide1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory upregulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

Excessive ammonium assimilation by plastidic glutamine synthetase causes ammonium toxicity in Arabidopsis thaliana.

Plants use nitrate, ammonium, and organic nitrogen in the soil as nitrogen sources. Since the elevated CO2 environment predicted for the near future will reduce nitrate utilization by C3 species, ammonium is attracting great interest. However, abundant ammonium nutrition impairs growth, i.e., ammonium toxicity, the primary cause of which remains to be determined. Here, we show that ammonium assimilation by GLUTAMINE SYNTHETASE 2 (GLN2) localized in the plastid rather than ammonium accumulation is a primary cause for toxicity, which challenges the textbook knowledge. With exposure to toxic levels of ammonium, the shoot GLN2 reaction produced an abundance of protons within cells, thereby elevating shoot acidity and stimulating expression of acidic stress-responsive genes. Application of an alkaline ammonia solution to the ammonium medium efficiently alleviated the ammonium toxicity with a concomitant reduction in shoot acidity. Consequently, we conclude that a primary cause of ammonium toxicity is acidic stress.

Nitrogen Nutrition Promotes Rhizome Bud Outgrowth via Regulation of Cytokinin Biosynthesis Genes and an Oryza longistaminata Ortholog of FINE CULM 1.

Oryza longistaminata, a wild rice, can propagate vegetatively via rhizome formation and, thereby, expand its territory through horizontal growth of branched rhizomes. The structural features of rhizomes are similar to those of aerial stems; however, the physiological roles of the two organs are different. Nitrogen nutrition is presumed to be linked to the vegetative propagation activity of rhizomes, but the regulation of rhizome growth in response to nitrogen nutrition and the underlying biological processes have not been well characterized. In this study, we analyzed rhizome axillary bud growth in response to nitrogen nutrition and examined the involvement of cytokinin-mediated regulation in the promotion of bud outgrowth in O. longistaminata. Our results showed that nitrogen nutrition sufficiency promoted rhizome bud outgrowth to form secondary rhizomes. In early stages of the response to nitrogen application, glutamine accumulated rapidly, two cytokinin biosynthesis genes, isopentenyltransferase, and CYP735A, were up-regulated with accompanying cytokinin accumulation, and expression of an ortholog of FINE CULM1, a negative regulator of axillary bud outgrowth, was severely repressed in rhizomes. These results suggest that, despite differences in physiological roles of these organs, the nitrogen-dependent outgrowth of rhizome axillary buds in O. longistaminata is regulated by a mechanism similar to that of shoot axillary buds in O. sativa. Our findings provide a clue for understanding how branched rhizome growth is regulated to enhance nutrient acquisition strategies.

Temperature-dependent fasciation mutants provide a link between mitochondrial RNA processing and lateral root morphogenesis.

Although mechanisms that activate organogenesis in plants are well established, much less is known about the subsequent fine-tuning of cell proliferation, which is crucial for creating properly structured and sized organs. Here we show, through analysis of temperature-dependent fasciation (TDF) mutants of Arabidopsis, root redifferentiation defective 1 (rrd1), rrd2, and root initiation defective 4 (rid4), that mitochondrial RNA processing is required for limiting cell division during early lateral root (LR) organogenesis. These mutants formed abnormally broadened (i.e. fasciated) LRs under high-temperature conditions due to extra cell division. All TDF proteins localized to mitochondria, where they were found to participate in RNA processing: RRD1 in mRNA deadenylation, and RRD2 and RID4 in mRNA editing. Further analysis suggested that LR fasciation in the TDF mutants is triggered by reactive oxygen species generation caused by defective mitochondrial respiration. Our findings provide novel clues for the physiological significance of mitochondrial activities in plant organogenesis.

Expression analysis of genes encoding malectin-like domain (MLD)- and leucine-rich repeat (LRR)- containing proteins in Arabidopsis thaliana.

Malectin is a maltose-binding endoplasmic reticulum protein conserved in animals. In Arabidopsis thaliana, we identified four genes that encode malectin-like domain (MLD)- and leucine-rich repeat (LRR)-containing proteins (AtMLLRs): two were receptor-like proteins (AtMLLR1 and 2) and the other two were extracellular proteins (AtMLLR3 and 4). The promoter:G3GFP+promoter:GUS assay indicated the organ- and cell-specific expression of the AtMLLR2 and AtMLLR3 genes.Abbreviations: Cmr: chloramphenicol-resistance marker; G3GFP: G3 green fluorescent protein; GUS: ß-glucuronidase; KD: kinase domain; LRR: leucine-rich repeat; MLD: malectin-like domain; RLK: receptor-like kinase; SP: signal peptide; TMD: transmembrane domain; Tnos: nopaline synthase terminator.

Simple Spectroscopic Determination of Nitrate, Nitrite, and Ammonium in Arabidopsis thaliana.

Plants use nitrate, nitrite, and ammonium as inorganic nitrogen (N) sources. These N compounds are included in plant tissues at various concentrations depending on the balance between their uptake and assimilation. Thus, the contents of nitrate, nitrite, and ammonium are physiological indicators of plant N economy. Here, we describe a protocol for measurement of these inorganic N species in A. thaliana shoots or roots.

Interactions between nitrate and ammonium in their uptake, allocation, assimilation, and signaling in plants.

Nitrogen (N) availability is a major factor determining plant growth and productivity. Plants acquire inorganic N from the soil, mainly in the form of nitrate and ammonium. To date, researchers have focused on these N sources, and demonstrated that plants exhibit elaborate responses at both physiological and morphological levels. Mixtures of nitrate and ammonium are beneficial in terms of plant growth, as compared to nitrate or ammonium alone, and therefore synergistic responses to both N sources are predicted at different steps ranging from acquisition to assimilation. In this review, we summarize interactions between nitrate and ammonium with respect to uptake, allocation, assimilation, and signaling. Given that cultivated land often contains both nitrate and ammonium, a better understanding of the synergism between these N sources should help to identify targets with the potential to improve crop productivity.

Arabidopsis Root-Type Ferredoxin:NADP(H) Oxidoreductase 2 is Involved in Detoxification of Nitrite in Roots.

Ferredoxin:NADP(H) oxidoreductase (FNR) plays a key role in redox metabolism in plastids. Whereas leaf FNR (LFNR) is required for photosynthesis, root FNR (RFNR) is believed to provide electrons to ferredoxin (Fd)-dependent enzymes, including nitrite reductase (NiR) and Fd-glutamine-oxoglutarate aminotransferase (Fd-GOGAT) in non-photosynthetic conditions. In some herbal species, however, most nitrate reductase activity is located in photosynthetic organs, and ammonium in roots is assimilated mainly by Fd-independent NADH-GOGAT. Therefore, RFNR might have a limited impact on N assimilation in roots grown with nitrate or ammonium nitrogen sources. AtRFNR genes are rapidly induced by application of toxic nitrite. Thus, we tested the hypothesis that RFNR could contribute to nitrite reduction in roots by comparing Arabidopsis thaliana seedlings of the wild type with loss-of-function mutants of RFNR2 When these seedlings were grown under nitrate, nitrite or ammonium, only nitrite nutrition caused impaired growth and nitrite accumulation in roots of rfnr2 Supplementation of nitrite with nitrate or ammonium as N sources did not restore the root growth in rfnr2 Also, a scavenger for nitric oxide (NO) could not effectively rescue the growth impairment. Thus, nitrite toxicity, rather than N depletion or nitrite-dependent NO production, probably causes the rfnr2 root growth defect. Our results strongly suggest that RFNR2 has a major role in reduction of toxic nitrite in roots. A specific set of genes related to nitrite reduction and the supply of reducing power responded to nitrite concomitantly, suggesting that the products of these genes act co-operatively with RFNR2 to reduce nitrite in roots.

Local Anesthetics and Antipsychotic Phenothiazines Interact Nonspecifically with Membranes and Inhibit Hexose Transporters in Yeast.

Action mechanisms of anesthetics remain unclear because of difficulty in explaining how structurally different anesthetics cause similar effects. In Saccharomyces cerevisiae, local anesthetics and antipsychotic phenothiazines induced responses similar to those caused by glucose starvation, and they eventually inhibited cell growth. These drugs inhibited glucose uptake, but additional glucose conferred resistance to their effects; hence, the primary action of the drugs is to cause glucose starvation. In hxt(0) strains with all hexose transporter (HXT) genes deleted, a strain harboring a single copy of HXT1 (HXT1s) was more sensitive to tetracaine than a strain harboring multiple copies (HXT1m), which indicates that quantitative reduction of HXT1 increases tetracaine sensitivity. However, additional glucose rather than the overexpression of HXT1/2 conferred tetracaine resistance to wild-type yeast; therefore, Hxts that actively transport hexoses apparently confer tetracaine resistance. Additional glucose alleviated sensitivity to local anesthetics and phenothiazines in the HXT1m strain but not the HXT1s strain; thus, the glucose-induced effects required a certain amount of Hxt1. At low concentrations, fluorescent phenothiazines were distributed in various membranes. At higher concentrations, they destroyed the membranes and thereby delocalized Hxt1-GFP from the plasma membrane, similar to local anesthetics. These results suggest that the aforementioned drugs affect various membrane targets via nonspecific interactions with membranes. However, the drugs preferentially inhibit the function of abundant Hxts, resulting in glucose starvation. When Hxts are scarce, this preference is lost, thereby mitigating the alleviation by additional glucose. These results provide a mechanism that explains how different compounds induce similar effects based on lipid theory.

Tetracaine, a local anesthetic, preferentially induces translational inhibition with processing body formation rather than phosphorylation of eIF2α in yeast.

It is unclear whether local anesthetics, such as tetracaine, and antipsychotics, such as phenothiazines, act on lipids or proteins. In Saccharomyces cerevisiae, these drugs inhibit growth, translation initiation, and actin polarization, and induce cell lysis at high concentrations. These activities are likely due to the cationic amphiphilic structure common to these agents. Although drug-induced translational inhibition is conserved in mammalian cells, other mechanisms, including the phosphorylation of eIF2α, a eukaryotic translational initiation factor, remain poorly understood. At a concentration of 10 mM, tetracaine rapidly inhibited translation initiation and lysed cells, whereas, at 2.5 mM, it slowly induced inhibition without lysis. The pat1 disruptant defective in mRNA decapping and the xrn1 disruptant defective in 5'-3' exoribonuclease were partially resistant to translational inhibition by tetracaine at each concentration, but the gcn2 disruptant defective in the eIF2α kinase was not. Phosphorylation of eIF2α was induced by 10 mM but not by 2.5 mM tetracaine, whereas processing bodies (P-bodies) were formed at 2.5 mM in Pat1-dependent and -independent manners. Therefore, administration of tetracaine inhibits translation initiation with P-body formation at both concentrations but acts via the Gcn2-eIF2α system only at the higher concentration. Because other local anesthetics and phenothiazines induced Pat1-dependent P-body formation, the mechanisms involved in translational inhibition by these cationic amphiphiles are similar. These results suggest that this dose-dependent biphasic translational inhibition by tetracaine results from an increase in membrane proteins that are indirectly inhibited by nonspecific interactions of cationic amphiphiles with membrane lipids.

Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins.

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

High CO2 triggers preferential root growth of Arabidopsis thaliana via two distinct systems under low pH and low N stresses.

Biomass allocation between shoots and roots is an important strategy used by plants to optimize growth in various environments. Root to shoot mass ratios typically increase in response to high CO2, a trend particularly evident under abiotic stress. We investigated this preferential root growth (PRG) in Arabidopsis thaliana plants cultivated under low pH/high CO2 or low nitrogen (N)/high CO2 conditions. Previous studies have suggested that changes in plant hormone, carbon (C) and N status may be related to PRG. We therefore examined the mechanisms underlying PRG by genetically modifying cytokinin (CK) levels, C and N status, and sugar signaling, performing sugar application experiments and determining primary metabolites, plant hormones and expression of related genes. Both low pH/high CO2 and low N/high CO2 stresses induced increases in lateral root (LR) number and led to high C/N ratios; however, under low pH/high CO2 conditions, large quantities of C were accumulated, whereas under low N/high CO2 conditions, N was severely depleted. Analyses of a CK-deficient mutant and a starchless mutant, in conjunction with sugar application experiments, revealed that these stresses induce PRG via different mechanisms. Metabolite and hormone profile analysis indicated that under low pH/high CO2 conditions, excess C accumulation may enhance LR number through the dual actions of increased auxin and decreased CKs.

Nitrate addition alleviates ammonium toxicity without lessening ammonium accumulation, organic acid depletion and inorganic cation depletion in Arabidopsis thaliana shoots.

When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.