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.

Nitrogen (N) deficiency reduces crop yield, but this effect may be mitigated by symbiotic interactions between crops and fungi. However, the response of wheat-fungal interactions to N deficiency remains unclear. We hypothesised that wheat cultivars with a higher reported nitrogen use efficiency (NUE), would induce shifts in the fungal community composition and functional profiles within the wheat rhizosphere to tolerate N deficiency. A glasshouse experiment was conducted to examine the effects of N deficiency on the rhizosphere fungal communities of wheat (Triticum aestivum L.) cultivars Gladius (low N-use efficiency) and Mace (high N-use efficiency). Plants were grown until the mid-anthesis stage in a Dermosol soil treated with either 0 (Low-N) or 90 kg N ha(-1) (High-N). The rhizosphere fungal communities were characterised using quantitative PCR, ITS rRNA metabarcoding, and metagenomics. The abundance and diversity of the rhizosphere fungal community were not significantly influenced by N deficiency in either Mace or Gladius cultivars (P > 0.05). However, the fungal community composition showed significant variation across N treatments in Mace (P < 0.05), whereas no such effect was observed in Gladius (P > 0.05). Differential abundance analysis and fungal trait predictions indicated a reduction in fungal symbionts in both cultivars under N deficiency (P < 0.05). Metagenomic analysis demonstrated that fungal functional profiles remained unaffected by N deficiency (P > 0.05) but significantly differed between Mace and Gladius (P < 0.05). This study reveals intraspecific variation in rhizosphere fungal responses to N deficiency between Mace and Gladius. The metabarcoding and metagenomic data suggest functional redundancy within the fungal community, which may enhance wheat resilience under N-deficient conditions. These findings highlight the potential of using fungal community stability in developing biofertiliser products for sustainable agriculture.

Polyamines are small, polycationic molecules with amino groups that are present in most living organisms. Studies indicate that polyamines increase general protein synthesis and are essential for efficient translation. While progress has been made in understanding the role of polyamines in translation in bacteria and mammals, their contribution and mode of action in plants remain largely unexplored. In a previous study, we found that putrescine (Put) and the pathogen-associated molecular pattern (PAMP) from bacterial flagellin (flg22) transcriptionally induced ribosome biogenesis in plants. Here we examined the impact of polyamines (Put and spermine, Spm) and flg22 on ribosome complex formation in Arabidopsis. Our results indicate that polyamines, flg22 and their combinations increase the abundance of actively translating polysomes. Riboproteomic analyses revealed that polyamines and flg22 trigger differential changes in the accumulation of ribosomal proteins, which are structurally confined in response to Put. Importantly, Put was found binding to non-translating and actively translating ribosomes, suggesting that this polyamine has a role in functional aspects of translation, such as stabilization and/or remodeling of polysomal complexes. Additional global proteomics analyses in polyamine biosynthesis mutants revealed that lower Put availability triggers changes in proteins associated with ribonucleoprotein complex binding and biogenesis. Overall, our findings highlight the effect of polyamines and flg22 on shaping the ribosomal protein composition of actively translating ribosomes in plants.

Heat stress poses a significant threat to crop productivity; however, the thermotolerance mechanisms in underutilised oilseed crops, such as chia ( Salvia hispanica L.), remain poorly understood. Despite the growing interest in chia as a rich source of ω-3 fatty acids, its molecular response to heat stress, particularly in vegetative tissues, has not been explored. We conducted transcriptomic and lipidomic profiling to examine how chia leaves respond to short-term (3 h) and prolonged (27 h) heat stress, followed by recovery under ambient conditions. Heat stress induced differential expression in over 20% of transcripts in chia leaves, with distinct patterns involving Ca²⁺ signalling, heat shock factors, and other biological pathways contributing to cellular homeostasis. Gene expression and lipid profiles in chia leaves responded dynamically to both short-term (3 h) and prolonged (27 h) heat stress (38°C/20°C). An almost complete return to baseline was observed, with all but 0.3% of heat-responsive genes reverting to control expression levels after 24 h. Our analysis confirms the role of Ca²⁺-mediated signalling pathways and molecular chaperones, including heat shock proteins and heat shock factors, which have been previously shown to contribute to maintaining cellular function during heat stress in other plant species. Among 287 annotated lipid species, TGs exhibited the most significant and reversible changes (>2-fold), suggesting their involvement in membrane remodelling. Our findings reveal adaptive mechanisms in chia that may open avenues for enhancing thermotolerance in other heat-sensitive oilseed crops. SIGNIFICANCE STATEMENT Understanding how crops respond to heat stress is crucial as global temperatures rise. This study indicates that in chia ( Salvia hispanica ), pathways responding to heat stress, like calcium signalling and heat shock proteins, are rapidly activated, and leaf triacylglycerol levels rise under heat stress before returning to baseline during recovery. By demonstrating near-complete recovery of gene expression following heat exposure, these findings highlight mechanisms of thermotolerance that may support improved stress tolerance in other crops. ### Competing Interest Statement The authors have declared no competing interest. The Australian Government, Australian Research Training Program Scholarship University of Melbourne, https://ror.org/01ej9dk98, Alfred Nicholas Fellowship, Megan Klemm Postgraduate Research Scholarship, Norma Hilda Schuster (nee Swift) Scholarship, Botany Foundation Fellowship Award Rosewood Research

Plant acclimation occurs through system-wide mechanisms that include proteome shifts, some of which occur at the level of protein synthesis. All proteins are synthesized by ribosomes. Rather than being monolithic, transcript-to-protein translation machines, ribosomes can be selective and cause proteome shifts. In this study, we use apical root meristems of germinating seedlings of the monocotyledonous plant barley as a model to examine changes in protein abundance and synthesis during cold acclimation. We measured metabolic and physiological parameters that allowed us to compare protein synthesis in the cold to optimal rearing temperatures. We demonstrated that the synthesis and assembly of ribosomal proteins are independent processes in root proliferative tissue. We report the synthesis and accumulation of various macromolecular complexes and propose how ribosome compositional shifts may be associated with functional proteome changes that are part of successful cold acclimation. Our study indicates that translation initiation is limiting during cold acclimation while the ribosome population is remodelled. The distribution of the triggered ribosomal protein heterogeneity suggests that altered compositions may confer 60S subunits selective association capabilities towards translation initiation complexes. To what extent selective translation depends on heterogeneous ribo-proteome compositions in barley proliferative root tissue remains a yet unresolved question.This article is part of the discussion meeting issue 'Ribosome diversity and its impact on protein synthesis, development and disease'.

Elevated atmospheric [CO2] (e[CO2]) may alleviate the effects of water stress on plants. It is unclear however whether this results exclusively from changes in stomatal conductance and water savings or also reflects changes in metabolic pathways triggered by the extra carbohydrate supplies under e[CO2]. To help address this knowledge gap, metabolite patterns were analysed in leaves and nodules of lentils grown in a Free-Air CO2 Enrichment facility in a water limited agro-ecosystem over the course of two contrasting growing seasons, one with high (well above average), and one with low (well below average) rainfall. Metabolomic analyses of tissues sampled at flowering showed contrasting responses to e[CO2] in the contrasting seasons. In the high rainfall season, e[CO2] was associated with more pronounced signatures of active energy and amino acid metabolism in leaves as well as in nodules, and particularly increased abundance of proteinogenic amino acids in leaves and nodules, which suggested strong stimulation of nodule N2-fixation and N supply to leaves. In the low rainfall season, e[CO2] was associated with high abundance of stress responsive metabolites, including putative osmoprotectants such as sugars and polyols as well as some N-containing compounds (proline, gamma-aminobutyric acid, putrescine), while the concentration of proteinogenic amino acids in leaves was reduced. In nodules, e[CO2] was linked to lower concentrations of sugars, polyols and most proteinogenic amino acids, along with higher concentrations of N-containing stress metabolites. However, there was little evidence that e[CO2] enhanced energy and amino acid metabolism in the low rainfall season. This study suggests that e[CO2] amplifies rather than mitigates the effect of different seasons on lentil metabolism. Whilst in a high rainfall season e[CO2] intensified metabolic patterns related to active growth and N-fixation, in a low rainfall season e[CO2] strengthened stress response signatures.

Salvia hispanica L. (chia) is a source of abundant ω-3 polyunsaturated fatty acids (ω-3-PUFAs) that are highly beneficial to human health. The genomic basis for this accrued ω-3-PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica. The highly contiguous 321.5-Mbp genome assembly covering all six chromosomes enabled the identification of 32,922 protein-coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein desaturase (ShSAD) gene family, is the main driver of the abundance of ω-3-PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help establish a molecular breeding target that can be leveraged through genome editing techniques to increase ω-3 content in oil crops.

Metabolomics is the study of identification and quantification of small molecules present in biological systems. Metabolomics analyses of biological samples involves getting a metabolic profile using analytical technologies and analyzing the data generated by these platforms to gain insight into the system's metabolic state. This article presents an overview of the software tools and methods used for the analysis, visualization and interpretation of metabolomics data. Specifically, this article details metabolomics workflows, metadata, standards in metabolomics, data processing and workflow softwares, metabolomics experiments repositories, metabolomics databases and statistical analysis methods for analyzing metabolomics data.

In this study, the effectiveness of preharvest fertigation using calcium thiosulfate (CaTS) and potassium thiosulfate (KTS) to enhance the shelf life of bell peppers was investigated. The nutrient solution was enriched with KTS (either 0.26 or 0.53 mM of a commercial formulation) or CaTS (0.66 mM of a commercial formulation), and compared to a commercial source-based nutrient solution as a control. Fruit quality attributes and the activity of key enzymes involved in oxidative stress defense, enzymatic browning, cell wall degradation, and membrane lipid degradation were investigated for 30 days of storage. Results showed that CaTS and KTS treatments preserved protein (a secondary energy reserve) and proline content, and enhanced the fruit's PSII activity throughout storage. Despite increased phenylalanine ammonia-lyase activity, CaTS and KTS fertigation did not affect polyphenol oxidase activity. CaTS and KTS fertigation increased the activity of five (out of seven) antioxidant enzymes and impaired the activity of one (out of two) cell wall hydrolytic enzyme. CaTS was the most effective treatment, followed by KTS (0.53 mM), in causing these promotive effects. In conclusion, preharvest application of CaTS (0.66 mM) and KTS (0.53 mM) prolonged the postharvest life and delayed senescence of bell pepper fruits by preserving fruit weight, firmness, and photosynthetic performance, as well as enhancing the antioxidant defense system.

Nitrogen fertilization in agriculture has serious environmental consequences, including production of the greenhouse gas nitrous oxide (N2O), pollution of groundwater with nitrate (NO3-), and river eutrophication. Nitrogen use efficiency can be increased by amending fertilizers with inhibitors to slow microbial nitrification processes, which transform ammonia to NO3-. Unfortunately, commercial inhibitors have failed to perform reliably across various agroecosystems for reasons not well understood. Using a combination of bacterial studies and soil incubations, we demonstrate here that 4-methyl-1-(prop-2-yn-1-yl)-1H-1,2,3-triazole (MPT) exhibits superior nitrification inhibitory properties. Unlike the commercial reversible inhibitors, MPT acts as a mechanistic, irreversible inhibitor of the key enzyme ammonia monooxygenase, enabling effective retention of ammonium (NH4+) and suppression of NO3- and N2O production over 21 days in several agricultural soils with pH values ranging from 4.7 to 7.5. A bacterial viability stain and a suite of freshwater and terrestrial ecotoxicity tests did not indicate any acute or chronic toxicity. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed an enhanced inhibitory effect of MPT on both ammonia-oxidizing bacteria and archaea. Thus, MPT outperforms currently available nitrification inhibitors and has great potential for broad application in various agricultural settings.

BACKGROUND:Chia (Salvia hispanica L.) seeds have become increasingly popular among health-conscious consumers owing to their high content of ω-3 fatty acids, which provide various health benefits. Comprehensive chemical analyses of the fatty acids and proteins in chia seeds have been conducted, revealing their functional properties. Recent studies have confirmed the high ω-3 content of chia seed oil and have hinted at additional functional characteristics. SCOPE:This review article aims to provide an overview of the botanical, morphological and biochemical features of chia plants, seeds and seed mucilage. Additionally, we discuss the recent developments in genetic and molecular research on chia, including the latest transcriptomic and functional studies that examine the genes responsible for chia fatty acid biosynthesis. In recent years, research on chia seeds has shifted its focus from studying the physicochemical characteristics and chemical composition of seeds to understanding the metabolic pathways and molecular mechanisms that contribute to their nutritional benefits. This has led to a growing interest in various pharmaceutical, nutraceutical and agricultural applications of chia. In this context, we discuss the latest research on chia and the questions that remain unanswered, and we identify areas that require further exploration. CONCLUSIONS:Nutraceutical compounds associated with significant health benefits, including ω-3 polyunsaturated fatty acids, proteins and phenolic compounds with antioxidant activity, have been measured in high quantities in chia seeds. However, comprehensive investigations through both in vitro experiments and in vivo animal and controlled human trials are expected to provide greater clarity on the medicinal, antimicrobial and antifungal effects of chia seeds. The recently published genome of chia and gene-editing technologies, such as CRISPR, facilitate functional studies deciphering molecular mechanisms of biosynthesis and metabolic pathways in this crop. This necessitates development of stable transformation protocols and creation of a publicly available lipid database, mutant collection and large-scale transcriptomic datasets for chia.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r -proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context -dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context -dependent functionality of r -protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to highresolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot -gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r -proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r -proteins and normalize their intensities by the sum of r -protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r -protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r -proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r -proteins determined by stable isotope -assisted mass spectrometry. Our method purifies the r -proteins in a reversibly denatured state, which offers the possibility for combined top -down and bottom -up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r -proteins. As such, our method is better suited to conserve post -translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r -proteins to select an appropriate protease for digestion. We avoid nonspecific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r -proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non -labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r -protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top -down and bottom -up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto highresolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements.

Background and Aims Nitrogen (N) deficiency in soil constrains plant growth, which may potentially be alleviated by beneficial soil microbes. However, there is limited knowledge of the plant-microbe interactions of wheat cultivars with different N-use efficiency (NUE) under N deficiency. Methods We investigated the responses of soil and root endosphere microorganisms as well as root metabolites of two wheat cultivars (cv. Mace and Gladius) with reported high and low NUE, respectively, using a glasshouse experiment and a hydroponic experiment with three N levels. Results The rhizosphere bacterial community of Mace shifted under N deficiency, but not in its root endosphere. Conversely, the rhizosphere bacterial community of Gladius remained unchanged under N deficiency but shifted in its root endosphere. The metagenomic analysis illustrated that bacterial growth and motility in the rhizosphere of Mace, but not of Gladius, were potentially enhanced under N deficiency. A 4-fold increase in octadecanoic acid in the root extract of Mace, but not Gladius, under N deficiency, suggesting the potential role of octadecanoic acid in shaping the rhizobacterial community in Mace with higher reported NUE. Conclusion Our study provides new evidence highlighting the divergent responses of wheat-associated microorganisms and root metabolites to N deficiency in the two cultivars. In addition to this, our findings suggest that wheat cultivars with higher NUE may selectively recruit beneficial bacterial communities through secreting specific metabolites, thereby enhancing their growth under N-limited conditions.

Biofertilisers comprised of plant growth promoting bacteria (PGPB) present a promising sustainable alternative to synthetic fertilisers. Bacteria which consistently colonise roots of specific plants across distinct environments, known as that plant’s core root microbiome, are particularly promising due to their colonisation competency. However, traditional, culture-based techniques can overlook promising PGPB which do not display commonly screened for plant growth promoting traits. Although numerous studies have isolated beneficial root bacteria, few have combined bacterial metabarcoding with culture-based techniques to identify novel biofertiliser candidates. In a two-pronged approach, 16S rRNA amplicon sequencing was used to define the core root microbiome of the model cereal plant, Brachypodium distachyon, grown in four distinct soils. From 7,042 amplicon sequence variants (ASVs) detected in root fractions, only 40 ASVs were common at a prevalence of 80

Background: Specialised anti-herbivory metabolites are abundant in the solanaceous genus Nicotiana. These metabolites include the large family of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). Many HGL-DTGs occur exclusively within the Nicotiana genus, but information from the molecular model species N. tabacum, N. benthamiana, and the tree tobacco N. glauca is limited. Objectives: We studied HGL-DTG occurrence and complexity in these species with the aim of providing in-depth reference annotations and comprehensive HGL-DTG inventories. Methods: We analysed polar metabolite extracts in comparison to the previously investigated wild reference species N. attenuata using positive ESI(+) and negative ESI(-) mode electrospray ionisation LC-MS and MS/MS. Results: We provide annotations of 66 HGL-DTGs with in-source and MS/MS fragmentation spectra for selected HGL-DTGs with exemplary fragment interpretations of ESI(+) as well as less studied ESI(-) spectra. We assemble a potential biosynthesis pathway comparing the presence of HGL-DTGs in N. tabacum, N. glauca, and N. benthamiana to N. attenuata. Approximately one-third of HGL-DTGs are chromatographically resolved isomers of hexose, deoxyhexose, or malonate conjugates. The number of isomers is especially high for conjugates with low numbers of deoxyhexose moieties. Conclusions: We extend the number of known HGL-DTGs with a focus on Nicotiana model species and demonstrate that the HGL-DTG family of N. tabacum plants can be surprisingly complex. Our study provides an improved basis with detailed references to previous studies of wild Nicotiana species and enables inference of HGL-DTG pathways with required enzymes for the biosynthesis of this important family of specialised defence metabolites.

Increases in soil salinity impact growth and agricultural yield by inhibiting plant functions. Interactions of fungal endophytes with crop plants can improve tolerance and is a less expensive approach. Here, the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) has been investigated. Metabolomics using GC-MS for polar metabolites and LC-MS for lipids was employed to provide insights into the biochemical changes in inoculated roots during the early stages of interaction. T. harzianum improved root growth of both genotypes in saline conditions whereas uninoculated roots were significantly shorter after salt treatment. The fungus reduced relative concentration of sugars in both genotypes under saline conditions but there was no change in organic acids. Amino acids decreased only in cv. Gairdner in fungus-inoculated roots under saline conditions. Lipid analysis suggested that salt stress causes large changes in roots but that inoculation with fungus greatly reduces the extent of these changes. By studying a tolerant and a sensitive genotype and their responses to salt and inoculation we have been able to develop hypotheses that explain the tolerance of Vlamingh to salt and how fungal inoculation changes the response of Gairdner to improve its tolerance.

Recently, 1,4-disubstituted 1,2,3-triazoles were reported by us as a new class of nitrification inhibitors, which can outperform the commercial compound 3,4-dimethylpyrazole phosphate (DMPP) in soil incubations. In this work, the mechanism of inhibition of five 1,2,3-triazoles with different substitution patterns was explored using a bacterial assay based on the measurement of nitrite (NO2-) production by pure cell cultures of Nitrosomonas europaea and Nitrosospira multiformis. While polar functional groups, such as amines, esters, and alkoxy residues, were detrimental to inhibiting production of NO2-, triazoles carrying only aliphatic substituents showed the highest inhibition of up to 98%. The observed correlation between lipophilicity and inhibitory activity suggests that more lipophilic compounds could more easily access the membrane-bound ammonia monooxygenase (AMO), which catalyzes the first step of the nitrification process. Measurement of the Michaelis-Menten kinetics suggests that the disubstituted 1,2,3-triazoles studied in this work act as reversible, noncompetitive inhibitors. Real-time measurements of the oxygen (O-2) consumption showed that the O-2 uptake rate by AMO follows zero-order kinetics in the presence of the triazoles, confirming the nonmechanistic mode of inhibition.

ABSTRACT A high-quality chromosome-level reference genome of S. hispanica was assembled and analysed. Ancestral whole-genome duplication events have not promoted the high α-linolenic acid content in S. hispanica seeds Tandem duplication of six stearoyl-ACP desaturase genes is a plausible cause for high ω-3 content in chia seeds. Salvia hispanica L. (chia) is an abundant source of ω-3 polyunsaturated fatty acids (PUFAs) that are highly beneficial to human health. The genomic basis for this accrued PUFA content in this emerging crop was investigated through the assembly and comparative analysis of a chromosome-level reference genome for S. hispanica (321.5 Mbp). The highly contiguous 321.5Mbp genome assembly, which covers all six chromosomes enabled the identification of 32,922 protein coding genes. Two whole-genome duplications (WGD) events were identified in the S. hispanica lineage. However, these WGD events could not be linked to the high α-linolenic acid (ALA, ω-3) accumulation in S. hispanica seeds based on phylogenomics. Instead, our analysis supports the hypothesis that evolutionary expansion through tandem duplications of specific lipid gene families, particularly the stearoyl-acyl carrier protein (ACP) desaturase ( ShSAD ) gene family, is the main driver of the abundance of ω-3 PUFAs in S. hispanica seeds. The insights gained from the genomic analysis of S. hispanica will help leveraging advanced genome editing techniques and will greatly support breeding efforts for improving ω-3 content in other oil crops.

The metabolome is the biochemical basis of plant form and function, but we know little about its macroecological variation across the plant kingdom. Here, we used the plant functional trait concept to interpret leaf metabolome variation among 457 tropical and 339 temperate plant species. Distilling metabolite chemistry into five metabolic functional traits reveals that plants vary on two major axes of leaf metabolic specialization-a leaf chemical defense spectrum and an expression of leaf longevity. Axes are similar for tropical and temperate species, with many trait combinations being viable. However, metabolic traits vary orthogonally to life-history strategies described by widely used functional traits. The metabolome thus expands the functional trait concept by providing additional axes of metabolic specialization for examining plant form and function.