Diurnal switches in diazotrophic lifestyle increase nitrogen contribution to cereals.

Uncoupling of biological nitrogen fixation from ammonia assimilation is a prerequisite step for engineering ammonia excretion and improvement of plant-associative nitrogen fixation. In this study, we have identified an amino acid substitution in glutamine synthetase, which provides temperature sensitive biosynthesis of glutamine, the intracellular metabolic signal of the nitrogen status. As a consequence, negative feedback regulation of genes and enzymes subject to nitrogen regulation, including nitrogenase is thermally controlled, enabling ammonia excretion in engineered Escherichia coli and the plant-associated diazotroph Klebsiella oxytoca at 23 °C, but not at 30 °C. We demonstrate that this temperature profile can be exploited to provide diurnal oscillation of ammonia excretion when variant bacteria are used to inoculate cereal crops. We provide evidence that diurnal temperature variation improves nitrogen donation to the plant because the inoculant bacteria have the ability to recover and proliferate at higher temperatures during the daytime.

Finding Balance in Adversity: Nitrate Signaling as the Key to Plant Growth, Resilience, and Stress Response.

To effectively adapt to changing environments, plants must maintain a delicate balance between growth and resistance or tolerance to various stresses. Nitrate, a significant inorganic nitrogen source in soils, not only acts as an essential nutrient but also functions as a critical signaling molecule that regulates multiple aspects of plant growth and development. In recent years, substantial advancements have been made in understanding nitrate sensing, calcium-dependent nitrate signal transmission, and nitrate-induced transcriptional cascades. Mounting evidence suggests that the primary response to nitrate is influenced by environmental conditions, while nitrate availability plays a pivotal role in stress tolerance responses. Therefore, this review aims to provide an overview of the transcriptional and post-transcriptional regulation of key components in the nitrate signaling pathway, namely, NRT1.1, NLP7, and CIPK23, under abiotic stresses. Additionally, we discuss the specificity of nitrate sensing and signaling as well as the involvement of epigenetic regulators. A comprehensive understanding of the integration between nitrate signaling transduction and abiotic stress responses is crucial for developing future crops with enhanced nitrogen-use efficiency and heightened resilience.

CALCIUM-DEPENDENT PROTEIN KINASE 32-mediated phosphorylation is essential for the ammonium transport activity of AMT1;1 in Arabidopsis roots.

Protein kinase-mediated phosphorylation modulates the absorption of many nutrients in plants. CALCIUM-DEPENDENT PROTEIN KINASES (CPKs) are key players in plant signaling to translate calcium signals into diverse physiological responses. However, the regulatory role of CPKs in ammonium uptake remains largely unknown. Here, using methylammonium (MeA) toxicity screening, CPK32 was identified as a positive regulator of ammonium uptake in roots. CPK32 specifically interacted with AMMONIUM TRANSPORTER 1;1 (AMT1;1) and phosphorylated AMT1;1 at the non-conserved serine residue Ser450 in the C-terminal domain. Functional analysis in Xenopus oocytes showed that co-expression of CPK32 and AMT1;1 significantly enhanced the AMT1;1-mediated inward ammonium currents. In transgenic plants, the phosphomimic variant AMT1;1S450E, but not the non-phosphorylatable variant AMT1;1S450A, fully complemented the MeA insensitivity and restored high-affinity 15NH4+ uptake in both amt1;1 and cpk32 mutants. Moreover, in the CPK32 knockout background, AMT1;1 lost its ammonium transport activity entirely. These results indicate that CPK32 is a crucial positive regulator of ammonium uptake in roots and the ammonium transport activity of AMT1;1 is dependent on CPK32-mediated phosphorylation.