Determination of optimal NH4+/K + concentration and corresponding ratio critical for growth of tobacco seedlings in a hydroponic system.

Inherently, ammonium (NH4 +) is critical for plant growth; however, its toxicity suppresses potassium (K+) uptake and vice-versa. Hence, attaining a nutritional balance between these two ions (NH4 + and K+) becomes imperative for the growth of tobacco seedlings. Therefore, we conducted a 15-day experimental study on tobacco seedlings exposed to different concentrations (47 treatments) of NH4 +/K+ at different corresponding 12 ratios simultaneously in a hydroponic system. Our study aimed at establishing the optimal NH4 +-K+ concentration and the corresponding ratio required for optimal growth of different tobacco plant organs during the seedling stage. The controls were the baseline for comparison in this study. Plants with low or excessive NH4 +-K+ concentration had leaf chlorosis or dark greenish colouration, stunted whole plant part biomass, and thin roots. We found that adequate K+ supply is a pragmatic way to mitigate NH4 +-induced toxicity in tobacco plants. The optimal growth for tobacco leaf and root was attained at NH4 +-K+ concentrations 2-2 mM (ratio 1:1), whereas stem growth was optimal at NH4 +-K+ 1-2 mM (1:2). The study provided an insight into the right combination of NH4 +/K+ that could mitigate or prevent NH4 + or K+ stress in the tobacco seedlings.

Integrated comparative transcriptome and physiological analysis reveals the metabolic responses underlying genotype variations in NH4+ tolerance.

Several mechanisms have been proposed to explain NH4 + toxicity. However, the core information about the biochemical regulation of plants in response to NH4 + toxicity is still lacking. In this study, the tissue NH4 + concentration is an important factor contributing to variations in plant growth even under nitrate nutrition and NH4 + tolerance under ammonium nutrition. Furthermore, NH4 + led to the reprogramming of the transcriptional profile, as genes related to trehalose-6-phosphate and zeatin biosynthesis were downregulated, whereas genes related to nitrogen metabolism, camalexin, stilbenoid and phenylpropanoid biosynthesis were upregulated. Further analysis revealed that a large number of genes, which enriched in phenylpropanoid and stilbenoid biosynthesis, were uniquely upregulated in the NH4 +- tolerant ecotype Or-1. These results suggested that the NH4 +-tolerant ecotype showed a more intense response to NH4 + by activating defense processes and pathways. Importantly, the tolerant ecotype had a higher 15NH4 + uptake and nitrogen utilization efficiency, but lower NH4 +, indicating the tolerant ecotype maintained a low NH4 + level, mainly by promoting NH4 + assimilation rather than inhibiting NH4 + uptake. The carbon and nitrogen metabolism analysis revealed that the tolerant ecotype had a stronger carbon skeleton production capacity with higher levels of hexokinase, pyruvate kinase, and glutamate dehydrogenase activity to assimilate free NH4 +, Taken together, the results revealed the core mechanisms utilized by plants in response to NH4 +, which are consequently of ecological and agricultural importance.

WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone.

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.

NH4+ Toxicity, Which Is Mainly Determined by the High NH4+/K+ Ratio, Is Alleviated by CIPK23 in Arabidopsis.

Ammonium (NH4+) toxicity is always accompanied by ion imbalances, and NH4+ and potassium (K+) exhibit a competitive correlation in their uptake and transport processes. In Arabidopsis thaliana, the typical leaf chlorosis phenotype in the knockout mutant of calcineurin B-like interacting protein kinase 23 (CIPK23) is high-NH4+-dependent under low-K+ condition. However, the correlation of K+ and NH4+ in the occurrence of leaf chlorosis in the cipk23 mutant has not been deeply elucidated. Here, a modified hydroponic experimental system with different gradients of NH4+ and K+ was applied. Comparative treatments showed that NH4+ toxicity, which is triggered mainly by the high ratio of NH4+ to K+ (NH4+/K+ ≥ 10:1 for cipk23) but not by the absolute concentrations of the ions, results in leaf chlorosis. Under high NH4+/K+ ratios, CIPK23 is upregulated abundantly in leaves and roots, which efficiently reduces the leaf chlorosis by regulating the contents of NH4+ and K+ in plant shoots, while promoting the elongation of primary and lateral roots. Physiological data were obtained to further confirm the role CIPK23 in alleviating NH4+ toxicity. Taken all together, CIPK23 might function in different tissues to reduce stress-induced NH4+ toxicity associated with high NH4+/K+ ratios by regulating the NH4+-K+ balance in Arabidopsis.

Spatio-temporal dynamics in global rice gene expression (Oryza sativa L.) in response to high ammonium stress.

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.