Integrative transcriptomic and metabolomic analysis of D-leaf of seven pineapple varieties differing in N-P-K% contents

Background Pineapple (Ananas comosus L. Merr.) is the third most important tropical fruit in China. In other crops, farmers can easily judge the nutritional requirements from leaf color. However, concerning pineapple, it is difficult due to the variation in leaf color of the cultivated pineapple varieties. A detailed understanding of the mechanisms of nutrient transport, accumulation, and assimilation was targeted in this study. We explored the D-leaf nitrogen (N), phosphorus (P), and potassium (K) contents, transcriptome, and metabolome of seven pineapple varieties. Results Significantly higher N, P, and K% contents were observed in Bali, Caine, and Golden pineapple. The transcriptome sequencing of 21 libraries resulted in the identification of 14,310 differentially expressed genes in the D-leaves of seven pineapple varieties. Genes associated with N transport and assimilation in D-leaves of pineapple was possibly regulated by nitrate and ammonium transporters, and glutamate dehydrogenases play roles in N assimilation in arginine biosynthesis pathways. Photosynthesis and photosynthesis-antenna proteins pathways were also significantly regulated between the studied genotypes. Phosphate transporters and mitochondrial phosphate transporters were differentially regulated regarding inorganic P transport. WRKY, MYB, and bHLH transcription factors were possibly regulating the phosphate transporters. The observed varying contents of K% in the D-leaves was associated to the regulation of K+ transporters and channels under the influence of Ca2+ signaling. The UPLC-MS/MS analysis detected 873 metabolites which were mainly classified as flavonoids, lipids, and phenolic acids. Conclusions These findings provide a detailed insight into the N, P, K% contents in pineapple D-leaf and their transcriptomic and metabolomic signatures.

Drought, Salinity, and Low Nitrogen Differentially Affect the Growth and Nitrogen Metabolism of Sophora japonica (L.) in a Semi-Hydroponic Phenotyping Platform

Abiotic stresses, such as salinity, drought, and nutrient deficiency adversely affect nitrogen (N) uptake and assimilation in plants. However, the regulation of N metabolism and N pathway genes in Sophora japonica under abiotic stresses is unclear. Sophora japonica seedlings were subjected to drought (5% polyethylene glycol 6,000), salinity (75mM NaCl), or low N (0.01mM NH4NO3) for 3weeks in a semi-hydroponic phenotyping platform. Salinity and low N negatively affected plant growth, while drought promoted root growth and inhibited aboveground growth. The NH4+/NO3− ratio increased under all three treatments with the exception of a reduction in leaves under salinity. Drought significantly increased leaf NO2− concentrations. Nitrate reductase (NR) activity was unaltered or increased under stresses with the exception of a reduction in leaves under salinity. Drought enhanced ammonium assimilation with increased glutamate synthase (GOGAT) activity, although glutamine synthetase (GS) activity remained unchanged, whereas salinity and low N inhibited ammonium assimilation with decreased GS activity under salt stress and decreased GOGAT activity under low N treatment. Glutamate dehydrogenase (GDH) activity also changed dramatically under different stresses. Additionally, expression changes of genes involved in N reduction and assimilation were generally consistent with related enzyme activities. In roots, ammonium transporters, especially SjAMT1.1 and SjAMT2.1a, showed higher transcription under all three stresses; however, most nitrate transporters (NRTs) were upregulated under salinity but unchanged under drought. SjNRT2.4, SjNRT2.5, and SjNRT3.1 were highly induced by low N. These results indicate that N uptake and metabolism processes respond differently to drought, salinity, and low N conditions in S. japonica seedlings, possibly playing key roles in plant resistance to environmental stress.

The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK–Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

BZR1 Regulates Brassinosteroid-Mediated Activation of AMT1;2 in Rice

Although it is known that brassinosteroids (BRs) play pleiotropic roles in plant growth and development, their roles in plant nutrient uptake remain unknown. Here, we hypothesized that BRs directly regulate ammonium uptake by activating the expression of rice AMT1-type genes. Exogenous BR treatment upregulated both AMT1;1 and AMT1;2 expression, while this induction was impaired in the BR-receptor gene BRI1 mutant d61-1. We then focused on brassinazole-resistant 1 (BZR1), a central hub of the BR signaling pathway, demonstrating the important role of this signaling pathway in regulating AMT1 expression and rice roots NH4+ uptake. The results showed that BR-induced expression of AMT1;2 was suppressed in BZR1 RNAi plants but was increased in bzr1-D, a gain-of-function BZR1 mutant. Further EMSA and ChIP analyses showed that BZR1 bound directly to the BRRE motif located in the promoter region of AMT1;2. Moreover, cellular ammonium contents, 15NH4+ uptake, and the regulatory effect of methyl-ammonium on root growth are strongly dependent on the levels of BZR1. Overexpression lines of BRI1 and BZR1 and Genetic combination of them mutants showed that BZR1 activates AMT1;2 expression downstream of BRI1. In conclusion, the findings suggest that BRs regulation of NH4+ uptake in rice involves transcription regulation of ammonium transporters.

Taxon-specific shifts in bacterial and archaeal transcription of dissolved organic matter cycling genes in a stratified fjord

A considerable fraction of organic matter derived from photosynthesis in the euphotic zone settles into the ocean's interior, and under way is degraded by diverse microbial consortia that utilize a suite of extracellular enzymes and membrane transporters. Still, the molecular details that regulate carbon cycling across depths remain little explored. As stratification in fjords has made them attractive models to explore patterns in biological oceanography, we here analyzed bacterial and archaeal transcription in samples from five depth layers in the Gullmar Fjord, Sweden. Transcriptional variation over depth correlated with gradients in chlorophyll a and nutrient concentrations. Differences in transcription between sampling dates (summer and early autumn), were strongly correlated with ammonium concentrations, which potentially was linked with a stronger influence of (micro-)zooplankton grazing in summer. Transcriptional investment in carbohydrate-active enzymes (CAZymes) decreased with depth and shifted toward peptidases, partly a result of elevated CAZyme transcription by Flavobacteriales, Cellvibrionales and Synechococcales at 2-25 m and a dominance of peptidase transcription by Alteromonadales and Rhodobacterales from 50 m and down. In particular, CAZymes for chitin, laminarin, and glycogen were important. High levels of transcription of ammonium transporters by Thaumarchaeota at depth (up to 18% of total transcription), along with the genes for ammonia oxidation and CO2-fixation, indicated that chemolithoautotrophy contributed to the carbon flux in the fjord. The taxon-specific expression of functional genes for processing of the marine DOM pool and nutrients across depths emphasizes the importance of different microbial foraging mechanisms across spatiotemporal scales for shaping biogeochemical cycles.

High-affinity ammonium transport by Arabidopsis thaliana AMT1;4

In plants high affinity transport proteins mediate the essential transport of ammonium across membranes. In Arabidopsis thaliana six of these AMmonium Transporters (AMTs) are encoded by the genome. All of them show a unique expression pattern. While most AMTs are highly expressed in the root, AtAMT1;4 expression is limited to the pollen grains and the pollen tube. Here, we addressed the transport characteristics of AtAMT1;4 in the heterologous Xenopus laevis oocytes system. The transport saturated and showed high affinity for ammonium with a Km value lower than 10 µM. Based on our electrophysiological analysis, we classified AtAMT1;4 as a high affinity ammonium transporter.

Thymol ameliorates ammonium toxicity via repressing polyamine oxidase-derived hydrogen peroxide and modulating ammonium transporters in rice root

Background Ammonium is an indispensable nutrient for crop growth, but anoxic conditions or inappropriate fertilizer usage result in the increase in ammonium content in soil. Excessive ammonium causes phytotoxicity. Thymol is a kind of natural phenolic compound with anti-microbial properties. However, little is known about the role of thymol in modulating plant physiology. Here we find the novel role of thymol in protecting rice from ammonium toxicity. Results Thymol remarkably rescued rice seedlings growth from ammonium stress, which may resulted from the attenuation of reactive oxygen species (ROS) accumulation, oxidative injury, and cell death in both shoots and roots. Polyamine oxidase (PAO) metabolizes polyamines to produce ROS in plants in response to stress conditions. Thymol blocked ammonium-induced upregulation of a set of rice PAOs, which contributed to the decrease in ROS content. In rice seedlings upon ammonium stress, thymol downregulate the expression of ammonium transporters (AMT1;1 and AMT1;2); thymol upregulated the expression of calcineurin B-like interacting protein kinase 23 (CIPK23) and calcineurin B-like binding protein 1 (CBL1), two negative regulators of AMTs. This may help rice avoid ammonium overload in excessive ammonium environment. Correlation analysis indicated that PAOs, AMTs, and CBL1 were the targets of thymol in the detoxification of excessive ammonium. Conclusion Thymol facilitates rice tolerance against ammonium toxicity by decreasing PAO-derived ROS and modulating ammonium transporters. Such findings may be applicable in the modulation of nutrient acquisition during crop production. Graphical abstract

Abscisic-acid-dependent regulation of Arabidopsis thaliana ammonium transport relies on ABI1 control of CIPK23 and AMT1

SummaryAmmonium uptake at plant roots is regulated at the transcriptional, post-transcriptional and post-translational levels. Phosphorylation by the protein kinase CIPK23 transiently inactivates the ammonium transporters (AMT1s) but the phosphatases activating AMT1s remain unknown. Here, we have identified the PP2C phosphatase ABI1 as an activator of AMTs in Arabidopsis thaliana. We show that high external ammonium concentrations elevate the stress phytohormone abscisic acid (ABA) by de-glycosylation. Active ABA is sensed by ABI1-PYL complexes followed by the inactivation of ABI1 activating CIPK23. Under favourable growth conditions, ABI1 reduces AMT1 phosphorylation, both by binding and inactivating CIPK23, and by the direct dephosphorylation of AMT1s. Thus, ABI1 is a positive regulator of ammonium uptake, coupling nutrient acquisition to abiotic stress signalling. Elevated ABA reduces ammonium uptake during stress situations, such as ammonium toxicity, whereas ABI1 reactivates AMT1s under favourable growth conditions.

Feedback inhibition of AMT1 NH4+-transporters mediated by CIPK15 kinase

Background Ammonium (NH4+), a key nitrogen form, becomes toxic when it accumulates to high levels. Ammonium transporters (AMTs) are the key transporters responsible for NH4+ uptake. AMT activity is under allosteric feedback control, mediated by phosphorylation of a threonine in the cytosolic C-terminus (CCT). However, the kinases responsible for the NH4+-triggered phosphorylation remain unknown. Results In this study, a functional screen identified protein kinase CBL-Interacting Protein Kinase15 (CIPK15) as a negative regulator of AMT1;1 activity. CIPK15 was able to interact with several AMT1 paralogs at the plasma membrane. Analysis of AmTryoshka, an NH4+ transporter activity sensor for AMT1;3 in yeast, and a two-electrode-voltage-clamp (TEVC) of AMT1;1 in Xenopus oocytes showed that CIPK15 inhibits AMT activity. CIPK15 transcript levels increased when seedlings were exposed to elevated NH4+ levels. Notably, cipk15 knockout mutants showed higher 15NH4+ uptake and accumulated higher amounts of NH4+ compared to the wild-type. Consistently, cipk15 was hypersensitive to both NH4+ and methylammonium but not nitrate (NO3−). Conclusion Taken together, our data indicate that feedback inhibition of AMT1 activity is mediated by the protein kinase CIPK15 via phosphorylation of residues in the CCT to reduce NH4+-accumulation.