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Abscisic acid plays a positive role in regulating the OsSAPK9–OsbZIP20 pathway and elevating antioxidant and GS/GOGAT activities in rice to increase tolerance to high-NH4+ stress., Ammonium (NH4+) is one of the principal nitrogen (N) sources in soils, but is typically toxic already at intermediate concentrations. The phytohormone abscisic acid (ABA) plays a pivotal role in responses to environmental stresses. However, the role of ABA under high-NH4+ stress in rice (Oryza sativa L.) is only marginally understood. Here, we report that elevated NH4+ can significantly accelerate tissue ABA accumulation. Mutants with high (Osaba8ox) and low levels of ABA (Osphs3-1) exhibit elevated tolerance or sensitivity to high-NH4+ stress, respectively. Furthermore, ABA can decrease NH4+-induced oxidative damage and tissue NH4+ accumulation by enhancing antioxidant and glutamine synthetase (GS)/glutamate synthetasae (GOGAT) enzyme activities. Using RNA sequencing and quantitative real-time PCR approaches, we ascertain that two genes, OsSAPK9 and OsbZIP20, are induced both by high NH4+ and by ABA. Our data indicate that OsSAPK9 interacts with OsbZIP20, and can phosphorylate OsbZIP20 and activate its function. When OsSAPK9 or OsbZIP20 are knocked out in rice, ABA-mediated antioxidant and GS/GOGAT activity enhancement under high-NH4+ stress disappear, and the two mutants are more sensitive to high-NH4+ stress compared with their wild types. Taken together, our results suggest that ABA plays a positive role in regulating the OsSAPK9–OsbZIP20 pathway in rice to increase tolerance to high-NH4+ stress.

Ammonium (NH4 + ) is toxic to root growth in most plants, even at moderate concentrations. Transcriptional regulation is one of the most important mechanisms in the response of plants to NH4 + toxicity, but the nature of the involvement of transcription factors (TFs) in this regulation remains unclear. Here, RNA-seq analysis was performed on Arabidopsis roots to screen for ammonium-responsive TFs. WRKY46, the member of the WRKY transcription factor family most responsive to NH4 + , was selected. We defined the role of WRKY46 using mutation and overexpression assays, and characterized the regulation of NUDX9 and indole-3-acetic acid (IAA)-conjugating genes by WRKY46 via yeast one-hybrid and electrophoretic mobility shift assays and chromatin immunoprecipitation-quantitative real-time polymerase chain reaction (ChIP-qPCR). Knockout of WRKY46 increased, while overexpression of WRKY46 decreased, NH4 + -suppression of the primary root. WRKY46 is shown to directly bind to the promoters of the NUDX9 and IAA-conjugating genes (GH3.1, GH3.6, UGT75D1, UGT84B2) and to inhibit their transcription, thus positively regulating free IAA content and stabilizing protein N-glycosylation, leading to an inhibition of NH4 + efflux in the root elongation zone (EZ). We identify TF involvement in the regulation of NH4 + efflux in the EZ, and show that WRKY46 inhibits NH4 + efflux by negative regulation of NUDX9 and IAA-conjugating genes.

Abscisic acid (ABA) plays a key role in plant growth and development and during stress responses. Plants respond to ABA through recognition, signal transduction, and response cascades. The core ABA signaling pathway consists of ABA receptors (RCAR/PYL/PYRs), protein phosphatases (PP2Cs), kinases (SnRK2s), transcription factors and ion channel proteins. Protein phosphorylation plays a key role in this pathway. In the absence of ABA, PP2Cs inhibit SnRK2s activities by dephosphorylating SnRK2s. When ABA binds to RCAR/PYL/PYRs, the complex then binds to PP2Cs, resulting in inactivation of the PP2Cs and release of the SnRK2s, which then phosphorylate a series of substrates to activate ABA responses. Selective protein degradation by the ubiquitin–proteasome system also contributes to regulation of ABA homeostasis, transport, signaling, and desensitization. The small ubiquitin-like modifier (SUMO) enhances the stability of ABI5 but also inhibits its transcription. ABA-induced reactive nitrogen and oxygen species regulate multiple key components of the ABA signaling pathways through redox-induced modifications (REDOX), such as oxidation, nitration, and nitrosylation, forming a feedback regulation mechanism that precisely regulates ABA signaling. This review will detail the role of these post-translational modifications in the core ABA signaling pathway.

Ammonium (NH4+) inhibits primary root (PR) growth in most plant species when present even at moderate concentrations. Previous studies have shown that transport of indole-3-acetic acid (IAA) is critical to maintaining root elongation under high-NH4+ stress. However, the precise regulation of IAA homeostasis under high-NH4+ stress (HAS) remains unclear. In this study, qRT-PCR, RNA-seq, free IAA and IAA conjugate and PR elongation measurements were conducted in genetic mutants to investigate the role of IAA biosynthesis and conjugation under HAS. Our data clearly show that HAS decreases free IAA in roots by increasing IAA inactivation but does not decrease IAA biosynthesis, and that the IAA-conjugating genes GH3.1, GH3.2, GH3.3, GH3.4, and GH3.6 function as the key genes in regulating high-NH4+ sensitivity in the roots. Furthermore, the analysis of promoter::GUS staining in situ and genetic mutants reveals that HAS promotes IAA conjugation in the elongation zone (EZ), which may be responsible for the PR inhibition observed under HAS. This study provides potential new insight into the role of auxin in the improvement of tolerance to NH4+.

Ammonium (NH4+) is toxic to root growth in most plants already at moderate levels of supply, but mechanisms of root growth tolerance to NH4+ remain poorly understood. Here, we report that high levels of NH4+ induce nitric oxide (NO) accumulation, while inhibiting potassium (K+) acquisition via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4), leading to the arrest of primary root growth. High levels of NH4+ also stimulated the accumulation of GSNOR (S-nitrosoglutathione reductase) in roots. GSNOR overexpression improved root tolerance to NH4+. Loss of GSNOR further induced NO accumulation, increased SNO1/SOS4 activity, and reduced K+ levels in root tissue, enhancing root growth sensitivity to NH4+. Moreover, the GSNOR-like gene, OsGSNOR, is also required for NH4+ tolerance in rice. Immunoblotting showed that the NH4+-induced GSNOR protein accumulation was abolished in the VTC1- (vitamin C1) defective mutant vtc1-1, which is hypersensititive to NH4+ toxicity. GSNOR overexpression enhanced vtc1-1 root tolerance to NH4+. Our findings suggest that induction of GSNOR increases NH4+ tolerance in Arabidopsis roots by counteracting NO-mediated suppression of tissue K+, which depends on VTC1 function.

The auxin IAA is a vital plant hormone in controlling growth and development, but our knowledge about its complicated biosynthetic pathways and molecular regulation are still limited and fragmentary. cytokinin induced root waving 2 (ckrw2) was isolated as one of the auxin-deficient mutants in a large-scale forward genetic screen aiming to find more genes functioning in auxin homeostasis and/or its regulation. Here we show that CKRW2 is identical to Histone Monoubiquitination 1 (HUB1), a gene encoding an E3 ligase required for histone H2B monoubiquitination (H2Bub1) in Arabidopsis. In addition to pleiotropic defects in growth and development, loss of CKRW2/HUB1 function also led to typical auxin-deficient phenotypes in roots, which was associated with significantly lower expression levels of several functional auxin synthetic genes, namely TRP2/TSB1, WEI7/ASB1, YUC7 and AMI1. Corresponding defects in H2Bub1 were detected in the coding regions of these genes by chromatin immunoprecipitation (ChIP) analysis, indicating the involvement of H2Bub1 in regulating auxin biosynthesis. Importantly, application of exogenous cytokinin (CK) could stimulate CKRW2/HUB1 expression, providing an epigenetic avenue for CK to regulate the auxin homeostasis. Our results reveal a previously unknown mechanism for regulating auxin biosynthesis via HUB1/2-mediated H2Bub1 at the chromatin level., Li Zhang et al. characterize ckrw2, cytokinin-induced root waving 2, as a mutant form of HUB1 in Arabidopsis, the gene required for histone H2B monoubiquitination. This study implicates the involvement of H2Bub1 in regulating auxin biosynthesis.

Great progress has been achieved in developing hybrid "super rice" varieties in China. Understanding morphological root traits in super rice and the mechanisms of nitrogen acquisition by the root system are of fundamental importance to developing proper fertilisation and nutrient management practices in their production. The present study was designed to study morphological and physiological traits in hybrid super rice roots that are associated with nitrogen use efficiency (NUE). Two hybrid super rice varieties (Yongyou12, YY; Jiayou 6, JY) and one common variety (Xiushui 134, XS) with differing NUE were cultivated hydroponically, and morphological and physiological traits of seedling roots in response to varying nitrogen conditions were investigated. Our results show that the hybrid cultivars YY and JY exhibit larger root systems, arising from a maximisation of root tips and from longer roots without changes in root diameter. The cross-sectional proportion of aerenchyma was significantly higher in super rice roots. The larger root system of super hybrid rice contributed to higher N accumulation and resulted in higher N uptake efficiency. 15N (15NH4+) labeling results show that YY and JY had an enhanced capacity for ammonium (NH4+) uptake. Moreover, YY and JY were more tolerant to high NH4+ and showed reduced futile NH4+ efflux. NH4+ efflux in the root elongation zone, measured by Non-invasive Micro-test Technology, was significantly lower than in XS. Taken together, our results suggest that a longer root, a larger number of tips, a better developed aerenchyma, a higher capacity for N uptake, and reduced NH4+ efflux from roots are associated with higher NUE and growth performance in hybrid super rice.

Nitrate is the preferred form of nitrogen for most plants, acting both as a nutrient and a signaling molecule. However, the components and regulatory factors governing nitrate uptake in bread wheat (Triticum aestivum), one of the world’s most important crop species, have remained unclear, largely due to the complexity of its hexaploid genome. Here, based on recently released whole-genome information for bread wheat, the high-affinity nitrate transporter2 (NRT2) and the nitrate-assimilation-related (NAR) gene family are characterized. We show that abscisic acid (ABA)- Glc ester deconjugation is stimulated in bread wheat roots by nitrate resupply following nitrate withdrawal, leading to enhanced root-tissue ABA accumulation, and that this enhancement, in turn, affects the expression of root-type NRT2/NAR genes. TaANR1 is shown to regulate nitrate-mediated ABA accumulation by directly activating TaBG1, while TaWabi5 is involved in ABA-mediated NO(3)(−) induction of NRT2/NAR genes. Building on previous evidence establishing ABA involvement in the developmental response to high-nitrate stress, our study suggests that ABA also contributes to the optimization of nitrate uptake by regulating the expression of NRT2/NAR genes under limited nitrate supply, offering a new target for improvement of nitrate absorption in crops.

Ammonium (NH4+) is an important nitrogen source and is widely used as a fertilizer in agricultural systems. However, excess NH4+ inhibits root growth, and, subsequently, vegetative shoot growth and yield. This study examines whether auxin is involved in differential NH4+ tolerance in rice (Oryza sativa L.), and how auxin is regulated under high-NH4+ conditions in rice. An NH4+-sensitive (Kasalath, Kas) and an NH4+-insensitive (Koshihikari, Kos) rive cultivar were cultured hydroponically with or without exogenous indole-3-acetic acid (IAA) and auxin biosynthesis inhibitors. Root growth, root area, tissue IAA content, and transcription of genes involved in auxin biosynthesis, conjugation and degradation were determined. pDR5::GUS staining and auxin measurement show that high NH4+ can decrease free IAA content in roots. In addition, quantitative RT-PCR, pharmacology, and genetics analysis suggest that Kos possesses a higher capacity for auxin biosynthesis and a weaker capacity for auxin metabolism compared to Kas under high-NH4+ stress. We conclude that the NH4+-tolerant cultivar possesses a higher capacity to maintain auxin homeostasis under high-NH4+ stress, and that this advantage is incurred by promotion of auxin biosynthesis and a suppression of auxin metabolism.

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the a-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.

We identified a novel Arabidopsis ammonium tolerance 1 (amot1) mutant and reveal that blocking of ethylene signaling reduces tissue H2O2 accumulation and enhances seedling tolerance to ammonium stress., Ammonium (NH4+) toxicity inhibits shoot growth in Arabidopsis, but the underlying mechanisms remain poorly characterized. Here, we show that a novel Arabidopsis mutant, ammonium tolerance 1 (amot1), exhibits enhanced shoot growth tolerance to NH4+. Molecular cloning revealed that amot1 is a new allele of EIN3, a key regulator of ethylene responses. The amot1 mutant and the allelic ein3-1 mutants show greater NH4+ tolerance than the wild type. Moreover, transgenic plants overexpressing EIN3 (EIN3ox) are more sensitive to NH4+ toxicity The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) increases shoot sensitivity to NH4+, whereas the ethylene perception inhibitor Ag+ decreases sensitivity. NH4+ induces ACC and ethylene accumulation. Furthermore, ethylene-insensitive mutants such as etr1-3 and ein3eil1 display enhanced NH4+ tolerance. In contrast, the ethylene overproduction mutant eto1-1 exhibits decreased ammonium tolerance. AMOT1/EIN3 positively regulates shoot ROS accumulation, leading to oxidative stress under NH4+ stress, a trait that may be related to increased expression of peroxidase-encoding genes. These findings demonstrate the role of AMOT1/EIN3 in NH4+ tolerance and confirm the strong link between NH4+ toxicity symptoms and the accumulation of hydrogen peroxide.

Tamarix ramosissima Ledeb., a major host plant for the parasitic angiosperm Cistanche tubulosa, and known for its unique drought tolerance, has significant ecological and economic benefits. However, the mechanisms of nitrogen acquisition by the T. ramosissima root system under drought have remained uncharacterized. Here, uptake of nitrate (NO3-) in various regions of the root system was measured in T. ramosissima using Non-invasive Micro-test Technology at the cellular level, and using a 15NO3--enrichment technique at the whole-root level. These results were compared with responses in the model system cotton (Gossypium hirsutum L.). Tamarix ramosissima had lower net NO3- influx and a significantly lower Km (the apparent Michalis-Menten constant; 8.5 μM) for NO3- uptake than cotton under normal conditions. Upon simulated drought conditions, using polyethylene glycol (PEG), NO3- flux in cotton switched from net influx to net efflux, with a substantive peak in the white zone (WZ) of the root. There were no significant NO3- influx signals observed in the WZ of T. ramosissima under control conditions, whereas PEG treatment significantly enhanced NO3- influx in the WZ of T. ramosissima. The effect of PEG application on NO3- fluxes was highly localized, and the increase in net NO3- influx in response to PEG stimulation was also found in C. tubulosa-inoculated T. ramosissima. Consistently, root nitrogen (N) content and root biomass were higher in T. ramosissima than in cotton under PEG treatment. Our study provides insights into NO3- uptake and the influence of C. tubulosa inoculation in T. ramosissima roots during acclimation to PEG-induced drought stress and provides guidelines for silvicultural practice and for breeding of T. ramosissima under coupled conditions of soil drought and N deficiency.

The root tip zone is regarded as the principal action site for iron (Fe) toxicity and is more sensitive than other root zones, but the mechanism underpinning this remains largely unknown. We explored the mechanism underpinning the higher sensitivity at the Arabidopsis root tip and elucidated the role of nitric oxide (NO) using NO-related mutants and pharmacological methods. Higher Fe sensitivity of the root tip is associated with reduced potassium (K+ ) retention. NO in root tips is increased significantly above levels elsewhere in the root and is involved in the arrest of primary root tip zone growth under excess Fe, at least in part related to NO-induced K+ loss via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4) and reduced root tip zone cell viability. Moreover, ethylene can antagonize excess Fe-inhibited root growth and K+ efflux, in part by the control of root tip NO levels. We conclude that excess Fe attenuates root growth by effecting an increase in root tip zone NO, and that this attenuation is related to NO-mediated alterations in K+ homeostasis, partly via SNO1/SOS4.

The plant auxin indole-3-acetic acid (IAA) plays critical roles in plant growth and development. There are two main strategies proposed for plant synthesis of IAA: the Trp-dependent (TD) and the Trp-independent (TI) pathways. Four TD pathways, namely the indole-3-acetamide pathway, the indole-3-pyruvic acid pathway, the tryptamine pathway and the indole-3-acetaldoxime pathway, have been postulated, identified and extensively studied. On the other hand, neither genes nor mutants involved in the TI pathway have been identified to date. Interestingly, some bacteria have auxin synthesis pathways that are similar to those in plants, indicating conserved biosynthetic mechanisms. Over the past few years, genetic, biochemical and molecular studies have greatly advanced our understanding of auxin biosynthesis. This review both summarizes recent advances in genetic and molecular knowledge and addresses the unsolved questions regarding auxin biosynthesis pathways in plants.

Auxin is a major phytohormone involved in most aspects of plant growth and development. Generally, auxin is perceived by three distinct receptors: TRANSPORT INHIBITOR RESISTANT1-Auxin/INDOLE ACETIC ACID, S-Phase Kinase-Associated Protein 2A and AUXIN-BINDING PROTEIN1. The auxin perception is regulated by a variety of secondary messenger molecules, including nitric oxide, reactive oxygen species, calcium, cyclic GMP, cyclic AMP, inositol triphosphate, diacylglycerol and by physiological pH. In addition, some small organic molecules, including inositol hexakisphosphate, yokonolide B, p-chlorophenoxyisobutyric acid, toyocamycin and terfestatin A, are involved in auxin signaling. In this review, we summarize and discuss the recent progress in understanding the functions of these secondary messengers and small organic molecules, which are now thoroughly demonstrated to be pervasive and important in auxin perception and signal transduction.

T-DNA insertional mutagenesis is a powerful tool in Arabidopsis functional genomics research. Previous studies have developed thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) as an efficient strategy in isolation of DNA sequences adjacent to known sequences in T-DNA tagged mutants. However, a number of problems are encountered when attempts are made to clone flanking sequences in T-DNA tagged mutants. Therefore, it is necessary to improve the efficiency of cloning mutagenesis. Here, we present the most frequent problems and provide an improved method to increase TAIL-PCR efficiency. Even then, it is not always possible to successfully obtain flanking sequences; in such cases, we recommend using high-throughput sequencing to determine the mutations.

Ammonium (NH4+) is the predominant nitrogen (N) source in many natural and agricultural ecosystems, including flooded rice fields. While rice is known as an NH4+-tolerant species, it nevertheless suffers NH4+ toxicity at elevated soil concentrations. NH4+ excess rapidly leads to the disturbance of various physiological processes that ultimately inhibit shoot and root growth. However, the global transcriptomic response to NH4+ stress in rice has not been examined. In this study, we mapped the spatio-temporal specificity of gene expression profiles in rice under excess NH4+ and the changes in gene expression in root and shoot at various time points by RNA-Seq (Quantification) using Illumina HiSeqTM 2000. By comparative analysis, 307 and 675 genes were found to be up-regulated after 4h and 12h of NH4+ exposure in the root, respectively. In the shoot, 167 genes were up-regulated at 4h, compared with 320 at 12h. According to KEGG analysis, up-regulated DEGs mainly participate in phenylpropanoid (such as flavonoid) and amino acid (such as proline, cysteine, and methionine) metabolism, which is believed to improve NH4+ stress tolerance through adjustment of energy metabolism in the shoot, while defense and signaling pathways, guiding whole-plant acclimation, play the leading role in the root. We furthermore critically assessed the roles of key phytohormones, and found abscisic acid (ABA) and ethylene (ET) to be the major regulatory molecules responding to excess NH4+ and activating the MAPK (mitogen-activated protein kinase) signal-transduction pathway. Moreover, we found up-regulated hormone-associated genes are involved in regulating flavonoid biosynthesis and are regulated by tissue flavonoid accumulation.

Auxin and cytokinin (CK) are both important hormones involved in many aspects of plant growth and development. However, the details of auxin biosynthesis and the interaction between auxin and CK are still unclear. Isolation and characterization of an auxin deficient mutant cytokinin induced root curling 2 (ckrc2) in this work reveal that CKRC2 encodes a previously identified member of YUCCA (YUC) flavin monooxygenase-like proteins (YUC8). Our results show that, like other YUCs, CKRC2/YUC8 is a rate-limiting enzyme for catalyzing the conversion of indole-3-pyruvic acid (IPyA) to indole-3-acetic acid (IAA), acting downstream of CKRC1/TAA1 in the IPyA pathway. Here we show that the transcription of both CKRC1/TAA and CKRC2/YUC8 can be induced by CK and that the phytochrome-interacting factor 4 (PIF4) is required for this upregulation. Transcription of PIF4 itself is induced by CK via the AHKs-ARR1/12 signalling pathway. These results indicate that PIF4 plays an essential role in mediating the regulatory effect of CK on the transcriptions of CKRC1 and CKRC2 genes in the IPyA pathway of auxin biosynthesis.

Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.