Vegetable production systems are important sources of agricultural ammonia (NH3) emissions. However, the characteristics of NH3 emissions from global vegetable systems and effective mitigation measures remain poorly understood. This study conducted a meta-analysis to elucidate global NH3 emission patterns, drivers, and mitigation options in vegetable production. Additionally, we proposed sustainable integrated nitrogen (N) fertilizer management strategies and evaluated their effectiveness through two field trials. The meta-analysis showed that the global NH3 emission factors for open-field and greenhouse vegetable systems were 6.1% and 2.8%, respectively. The estimated global synthetic N fertilizer-induced NH3 emissions from vegetable systems were 1087 Gg N year−1, which was 46% lower than the estimate based on the Intergovernmental Panel on Climate Change default value. N fertilizer type, application rate, placement method, and soil mineral N concentration were identified as the primary predictors of N fertilizer-induced NH3 emissions. Furthermore, our meta-analysis demonstrated that knowledge-based 4 R nutrient stewardship practices effectively reduced NH3 emissions, particularly through optimized N application rates (−39%), use of slow/controlled release fertilizer (−52%), and subsurface application methods (−69%). Our field trials further demonstrated that integrated N management measures enhanced NH3 mitigation while increasing vegetable yield. This study provides quantitative evidence for developing effective NH3 mitigation strategies and advancing sustainable N management in global vegetable production systems.

Inositol phosphates and pyrophosphates are small, water-soluble molecules involved in a range of physiological processes across eukaryotic organisms, including plants. Over the past two decades, significant advancements in inositol (pyro)phosphate detection and chemical synthesis, coupled with the characterization of plant mutants and the structural analysis of receptors and associated proteins, have greatly enhanced our understanding of their production, degradation, and perception in plants. This growing knowledge base demonstrates that inositol (pyro)phosphates are crucial for regulating key processes, such as phosphorus homeostasis, hormone signaling, and plant-microbe interactions. We provide a global perspective on these processes, highlighting recent discoveries, new possibilities, and unresolved questions.

Inositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While certain PP-InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain-containing proteins in plants, the functions of the recently discovered isomer 4/6-PP-InsP(5) remain largely unknown. Here, we employ two complementary affinity-based strategies-a matrix approach and a photoaffinity probe-to systematically identify 4/6-PP-InsP(5)-binding proteins in Arabidopsis thaliana. The two methods yield partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers are applied to obtain information about potential isomer-specific interactions. As a proof-of-concept, one candidate interactor (FHA domain-containing protein AtFHA2) is shown to bind 4-PP-InsP(5) in vitro with markedly higher affinity than InsP(6), while no reliable binding parameters could be obtained for its enantiomer 6-PP-InsP(5). Thus, besides the SPX domain, FHA domain-containing proteins, of which 18 exist in Arabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings reveal a diverse protein network associated with 4/6-PP-InsP(5) and establish a versatile platform for dissecting its biological roles in plants and other organisms.

Plant growth-promoting bacteria can enhance plant performance under nutrient limitation, yet the underlying plant molecular responses remain incompletely resolved. We investigated growth promotion by Pseudomonas koreensis in Brachypodium distachyon under contrasting nitrogen (N) regimes using time-resolved phenotyping, elemental analysis, lipidomics and proteomics. Shoot phenotyping revealed rapid responses to N availability, whereas beneficial effects of bacterial inoculation emerged only during prolonged growth under low N. Under N limitation, inoculated plants accumulated significantly more biomass and total N than uninoculated controls, reaching levels comparable to high N plants, while no inoculation effect was observed under high N. Biomass increases were accompanied by only modest changes in tissue N concentration, indicating enhanced whole-plant N-use efficiency rather than disproportionate N enrichment. Proteomics identified N availability as the primary determinant of proteome structure, with bacterial inoculation under low N conditionally modulating selected modules towards High N states. Lipidomic profiles were largely N-driven, with only transient inoculation effects at early stages. Despite the presence of N fixation-associated genes in P. koreensis, δ15N analyses did not support substantial in planta N fixation. Together, these results support a plant-centric model in which bacterial inoculation enhances growth under N limitation by modulating plant-encoded N acquisition and metabolic organization within an N-defined framework.

Plant Cullin RING Ubiquitin E3 ligases (CRLs) play a critical role in targeted protein degradation, essential for physiological development and stress adaptation. The deneddylase activity of the COP9 signalosome (CSN) tightly regulates the cellular balance of neddylated cullins, which is crucial for maintaining the full spectrum of CRL functions. Although selective inositol polyphosphates (InsPs) act as cofactors in plant responses that involve ubiquitylation of negative regulators, their connection to CSN-CRL activities has remained unclear. In this study, we reveal that the two Arabidopsis thaliana InsP-kinases, IPK1 and ITPK1, physically interact and orchestrate the metabolic regulation of the CSN holo-complex activity. Notably, ITPK1 deficiency lowers Nedd8 processing rates, elevates the cellular ratios of neddylated cullins, and disturbs the dissociation equilibrium of CSN5 and CUL1 from the holo-complex. These findings uncover a novel autoregulatory switch in CSN functions, governed by deneddylation activity. Furthermore, we demonstrate that the phosphate starvation response (PSR), induced in phosphate-limited wild-type plants and constitutively active in the InsP-kinase mutants, is partly regulated by reduced deneddylation rates, which affect the stability of SPX4, a key negative regulator of PSR. Pharmacological inhibition of cullin neddylation stabilizes SPX4 and impairs PSR, thereby linking CSN-CRL dynamics to phosphate sensing. Conversely, pharmacologically inhibiting CSN5 deneddylase activity causes wild-type plants to exhibit PSR phenotypes similar to those of the InsP-kinase mutants. Collectively, these results reveal that specific InsP-kinases are partly involved in modulating plant PSR by fine-tuning the coordination between CRL and CSN activities.

Phosphorylated myo-inositols (InsPs) are essential cytoplasmic signaling molecules, while their lipidated analogs (PtdInsPs) play a crucial role in membrane signaling. Stereoselective synthesis of these compounds has been achieved through various methods, predominantly using the meso compound myo-inositol as a starting material. However, phytate (InsP6), also a meso compound, is the most abundant inositol derivative in plants - far more prevalent than myo-inositol itself. Despite its abundance, phytate has been rarely used in synthetic strategies for accessing a variety of chiral inositol phosphates and their derivatives through selective dephosphorylations on a preparative scale. Here, we report gram-scale (stereo)selective dephosphorylations of phytate using phytases and demonstrate the application of these products in generating modified InsPs through a transient phosphitylation approach. Notably, the bacterial effector XopH efficiently desymmetrizes meso-phytate to yield enantiomerically pure 1-OH-InsP5. This transformation renders the 1-position accessible for further modifications, which, in biological systems, is where glycerolphosphate diesters are attached. By using selective dephosphorylations with phytases in concert with chemoselective telescoping reaction sequences, this approach greatly advances the stereoselective synthesis of inositol phosphates and their derivatives, such as glycerophosphoinositols, from abundant InsP6.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well-characterized exhaustively phosphorylated PP-InsPs, such as 5-PP-InsP5 (5-InsP7) and 1,5-(PP)2-InsP4 (1,5-InsP8), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5-PP-InsP4 isomers in enantiopure form and assigned their structures using ³¹P-NMR analysis in combination with a chiral solvating agent. Additionally, we developed ¹⁸O-labeled PP-InsP4 standards for quantitative mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the study of PP-InsP4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation-induced root-specific PP-InsP4 isomer does not match any 5-PP-InsP4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6-OH]-InsP5 and [3-OH]-InsP5 at the 5-position, while other InsP5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP-InsP4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP-InsP4, providing an entry point for future studies on their biochemical activity and their physiological roles.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated signaling molecules that regulate diverse cellular processes, including phosphate homeostasis and energy metabolism across species. Despite extensive research on well-characterized exhaustively phosphorylated PP-InsPs, such as 5-PP-InsP5 (5-InsP7) and 1,5-(PP)2-InsP4 (1,5-InsP8), the functional relevance of less abundant not fully phosphorylated isomers, remains largely unknown. In this study, we synthesized all unsymmetric 5-PP-InsP4 isomers in enantiopure form and assigned their structures using 31P-NMR analysis in combination with a chiral solvating agent. Additionally, we developed 18O-labeled PP-InsP4 standards for mass spectrometry in combination with capillary electrophoresis (CE-MS), enabling the assignment of PP-InsP4 in Arabidopsis thaliana under phosphate starvation. Our findings show that the previously detected, phosphate starvation-induced root-specific PP-InsP4 isomer does not match any 5-PP-InsP4 isomer, contrary to previous suggestions, thus indicating an alternative phosphorylation pattern. Enzyme assays further demonstrate that Arabidopsis ITPK1 selectively phosphorylates [6-OH]-InsP5 and [3-OH]-InsP5 at the 5-position, while other InsP5 isomers remain unchanged. This suggests that an unidentified enzymatic activity is involved in the formation of the elusive root PP-InsP4 species. Our study provides a comprehensive framework for the synthesis, analysis, and functional investigation of PP-InsP4, providing an entry point for future studies on their biochemical activity and their physiological roles.

Recent studies have established a key role of inositol pyrophosphates (PP-InsPs) in regulating phosphate (Pi) homeostasis across various organisms. In plants, PP-InsP levels are intricately linked to Pi status, with this reciprocal regulation being especially pronounced under fluctuating Pi conditions compared to stable nutrient sufficiency or deficiency. Here, we present a hydroponic cultivation protocol for Arabidopsis and rice, designed to simulate distinct and fluctuating Pi regimes. The composition of the nutrient solutions in these systems can be easily adjusted, enabling precise monitoring of changes in PP-InsP metabolism, under both steady-state and dynamic Pi conditions. Plants grown hydroponically under different Pi regimes can also be analyzed for phosphate starvation response (PSR) phenotypes and subjected to inositol phosphate (InsP)/PP-InsP extraction and quantification using methods detailed in other chapters of this special issue.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX-type hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high-performance liquid chromatography (SAX-HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg2+ dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4-InsP7, while Subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher order mutants of Subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wild-type (WT) and Subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

Zusammenfassung Inositolpyrophosphate (PP‐InsPs) sind hochphosphorylierte Signalmoleküle, die in verschiedenen Organismen zentrale zelluläre Prozesse wie die Phosphathomöostase und den Energiestoffwechsel regulieren. Während gut charakterisierte, vollständig phosphorylierte PP‐InsPs wie 5‐PP‐InsP 5 (5‐InsP 7 ) und 1,5‐(PP) 2 ‐InsP 4 (1,5‐InsP 8 ) intensiv untersucht wurden, ist bislang kaum etwas über die Funktion wenig häufiger, nur partiell phosphorylierter Isomere bekannt. In dieser Studie wurden alle unsymmetrischen 5‐PP‐InsP 4 ‐Isomere in enantiomeren‐reiner Form synthetisiert und mithilfe von 31 P‐NMR‐Spektroskopie in Kombination mit einem chiralen Solvatisierungsreagenz strukturell zugewiesen. Zusätzlich entwickelten wir 18 O‐markierte PP‐InsP 4 ‐Standards für CE‐MS‐Analysen (Kapillarelektrophorese gekoppelt mit Massenspektrometrie), die die Identifizierung eines PP‐InsP 4 ‐Isomers in Arabidopsis thaliana unter Phosphatmangelbedingungen ermöglichten. Unsere Ergebnisse zeigen, dass das unter Phosphatmangel beobachtete, wurzelspezifische PP‐InsP 4 ‐Isomer entgegen früherer Annahmen keinem der synthetisierten 5‐PP‐InsP 4 ‐Isomere entspricht. Dies weist auf ein alternatives Phosphorylierungsmuster hin. Enzymatische Untersuchungen belegen zudem, dass Arabidopsis ITPK1 bevorzugt [6‐OH]‐InsP 5 und [3‐OH]‐InsP 5 an der 5‐Position phosphoryliert, während andere InsP 5 ‐Isomere unverändert bleiben. Dies legt nahe, dass eine bislang unbekannte enzymatische Aktivität an der Biosynthese des bislang nicht zugewiesenen PP‐InsP 4 ‐Isomers beteiligt ist. Unsere Studie bietet ein umfassendes methodisches Fundament für die Synthese, Analyse und funktionelle Charakterisierung von PP‐InsP 4 und eröffnet neue Perspektiven für die Erforschung ihrer biochemischen Eigenschaften und physiologischen Funktionen.

In recent years, there has been substantial progress in the development of methods to analyze inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs). However, many of these techniques are labor- and cost-intensive and can usually only be carried out by laboratories specialized in InsPs/PP-InsPs analysis. In this chapter, we present a simple method that exploits the fact that phosphorylation and/or dephosphorylation of certain InsP/PP-InsP species induces the activation of promoters driving the expression of genes involved in phosphate starvation response (PSR). By linking PSR-inducible promoters to the RUBY reporter, which allows continuous and noninvasive visualization of promoter activation, we provide a new analytical tool to monitor enzymatic activities that alter PP-InsP levels, eliminating the need for complex biochemical analysis. We present a step-by-step protocol showing how simple coinfiltration of promoter-RUBY T-DNA constructs together with PP-InsP pyrophosphatase-encoding T-DNAs into Nicotiana benthamiana leaves enables evaluation of the enzymatic activity of expressed pyrophosphatases, and mention possible downstream analyses by methods described in other chapters of this special issue.

Heterosis, or hybrid vigor, describes the superior performance of F1 hybrids compared to parental inbreds. While soil microbiomes are proposed to influence heterosis, it remains unclear how heterotic plants shape their microbiomes and how interactions relate to stress responses. Here, we investigate the role of rhizosheath formation-the soil tightly adhering to roots-in maize heterosis under nitrogen deprivation. Across sterilization, inoculation, and transplantation experiments, hybrids develop larger rhizosheaths than inbreds, and rhizosheath size associates with biomass heterosis. Rhizosheath-enriched genus Massilia correlates with lateral root density, rhizosheath size, and growth. Untargeted metabolomics and flavone-deficient mutants reveal links between Massilia and flavonoid pathways, while growth promotion by Massilia can also occur independently of host flavones. Metagenomic analysis shows that larger rhizosheaths recruit microbial functions related to nutrient cycling and stress adaptation. These findings identify rhizosheath formation as an integrative trait associated with heterosis and a promising target for breeding resilient crops.

An early, non-invasive, and on-site detection of nutrient deficiencies is critical to enable timely actions to prevent major losses of crops caused by lack of nutrients. While acquiring labeled data is very expensive, collecting images from multiple views of a crop is straightforward. Despite its relevance for practical applications, unsupervised domain adaptation where multiple views are available for the labeled source domain as well as the unlabeled target domain is an unexplored research area. In this work, we thus propose an approach that leverages multiple camera views in the source and target domain for unsupervised domain adaptation. We evaluate the proposed approach on two nutrient deficiency datasets. The proposed method achieves state-of-the-art results on both datasets compared to other unsupervised domain adaptation methods. The dataset and source code are available at https://github.com/jh-yi/MV-Match.

The antagonistic interplay between phosphorus (P) and zinc (Zn) in plants is well established. However, the molecular mechanisms mediating those interactions as influenced by arbuscular mycorrhizal (AM) symbiosis remain unclear. We investigated Zn concentrations, root AM symbiosis, and transcriptome profiles of maize roots grown under field conditions upon different P levels. We also validated genotype-dependent P-Zn uptake in selected genotypes from a MAGIC population and conducted mycorrhizal inoculation experiments using mycorrhizal-defective mutant pht1;6 to elucidate the significance of AM symbiosis in P-Zn antagonism. Finally, we assessed how P supply affects Zn transporters and Zn uptake in extraradical hyphae within a three-compartment system. Elevated P levels led to a significant reduction in maize Zn concentration across the population, correlating with a marked decline in AM symbiosis, thus elucidating the P-Zn antagonism. We also identified ZmPht1;6 is crucial for AM symbiosis and confirmed that P-Zn antagonistic uptake is dependent on AM symbiosis. Moreover, we found that high P suppressed the expression of the fungal RiZRT1 and RiZnT1 genes, potentially impacting hyphal Zn uptake. We conclude that high P exerts systemic regulation over root and AM hyphae-mediated Zn uptake in maize. These findings hold implications for breeding Zn deficiency-tolerant maize varieties.

Both deficiency and toxicity of the micronutrient boron lead to severe reductions in crop yield. Despite this agricultural importance, the molecular basis underlying boron homeostasis in plants remains unclear. To identify molecular players involved in boron homeostasis in maize (Zea mays L.), we measured boron levels in the Goodman-Buckler association panel and performed genome-wide association studies. These analyses identified a benzoxazinless (bx) gene, bx3, involved in the biosynthesis of benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which are major defense compounds in maize. Genes involved in DIMBOA biosynthesis are all located in close proximity in the genome, and benzoxazinoid biosynthesis mutants, including bx3, are all DIMBOA deficient. We determined that leaves of the bx3 mutant have a greater boron concentration than those of B73 control plants, which corresponded with enhanced leaf tip necrosis, a phenotype associated with boron toxicity. By contrast, other DIMBOA-deficient maize mutants did not show altered boron levels or the leaf tip necrosis phenotype, suggesting that boron is not associated with DIMBOA. Instead, our analyses suggest that the accumulation of boron is linked to the benzoxazinoid intermediates indolin-2-one (ION) and 3-hydroxy-ION. Therefore, our results connect boron homeostasis to the benzoxazinoid plant defense pathway through bx3 and specific intermediates, rendering the benzoxazinoid biosynthesis pathway a potential target for crop improvement under inadequate boron conditions.

Inositol pyrophosphates (PP-InsPs) are energy-rich molecules harboring one or more diphosphate moieties. PP-InsPs are found in all eukaryotes evaluated and their functional versatility is reflected in the various cellular events in which they take part. These include, among others, insulin signaling and intracellular trafficking in mammals, as well as innate immunity and hormone and phosphate signaling in plants. The molecular mechanisms by which PP-InsPs exert such functions are proposed to rely on the allosteric regulation via direct binding to proteins, by competing with other ligands, or by protein pyrophosphorylation. The latter is the focus of this review, where we outline a historical perspective surrounding the first findings, almost 20 years ago, that certain proteins can be phosphorylated by PP-InsPs in vitro. Strikingly, in vitro phosphorylation occurs by an apparent enzyme-independent but Mg2+-dependent transfer of the β-phosphoryl group of an inositol pyrophosphate to an already phosphorylated serine residue at Glu/Asp-rich protein regions. Ribosome biogenesis, vesicle trafficking and transcription are among the cellular events suggested to be modulated by protein pyrophosphorylation in yeast and mammals. Here we discuss the latest efforts in identifying targets of protein pyrophosphorylation, pointing out the methodological challenges that have hindered the full understanding of this unique post-translational modification, and focusing on the latest advances in mass spectrometry that finally provided convincing evidence that PP-InsP-mediated pyrophosphorylation also occurs in vivo. We also speculate about the relevance of this post-translational modification in plants in a discussion centered around the protein kinase CK2, whose activity is critical for pyrophosphorylation of animal and yeast proteins. This enzyme is widely present in plant species and several of its functions overlap with those of PP-InsPs. Until now, there is virtually no data on pyrophosphorylation of plant proteins, which is an exciting field that remains to be explored.

Beneficial interactions with microorganisms are pivotal for crop performance and resilience. However, it remains unclear how heritable the microbiome is with respect to the host plant genotype and to what extent host genetic mechanisms can modulate plant–microbiota interactions in the face of environmental stresses. Here we surveyed 3,168 root and rhizosphere microbiome samples from 129 accessions of locally adapted Zea, sourced from diverse habitats and grown under control and different stress conditions. We quantified stress treatment and host genotype effects on the microbiome. Plant genotype and source environment were predictive of microbiome abundance. Genome-wide association analysis identified host genetic variants linked to both rhizosphere microbiome abundance and source environment. We identified transposon insertions in a candidate gene linked to both the abundance of a keystone bacterium Massilia in our controlled experiments and total soil nitrogen in the source environment. Isolation and controlled inoculation of Massilia alone can contribute to root development, whole-plant biomass production and adaptation to low nitrogen availability. We conclude that locally adapted maize varieties exert patterns of genetic control on their root and rhizosphere microbiomes that follow variation in their home environments, consistent with a role in tolerance to prevailing stress. He et al. found that locally adapted maize varieties exert patterns of genetic control on their root and rhizosphere microbiomes that follow variation in their home environments, consistent with a role in tolerance to prevailing stress.