Most plant genomes and their (post-)transcriptional regulation remain unknown. We used SPLASH-a new, reference genome-free sequence variation detection algorithm-to analyze transcriptional and post-transcriptional regulation from RNA-seq data. We discovered allelic variation in expression during maize pollen development and imbibition-dependent cryptic splicing in Arabidopsis seeds. SPLASH enables discovery of novel regulatory mechanisms, including differential regulation of genes from parental haplotypes of hybrids, without the use of alignment to a reference genome.

Global crop production faces increasing threats from the rise in frequency, duration, and intensity of drought and heat stress events due to climate change. Most staple food crops, including wheat, rice, soybean, and corn that provide over half of the world's caloric intake, are not well adapted to withstand heat or drought. Efforts to breed or engineer stress-tolerant crops have had limited success due to the complexity of tolerance mechanisms and the variability of agricultural environments. Effective solutions require a shift towards fundamental research that incorporates realistic agricultural settings and focuses on practical outcomes for farmers. This review explores the genetic and environmental factors affecting heat and drought tolerance in major crops, examines the physiological and molecular mechanisms underlying these stress responses, and evaluates the limitations of current breeding programs and models. It also discusses emerging technologies and approaches that could enhance crop resilience, such as synthetic biology, advanced breeding techniques, and high-throughput phenotyping. Finally, this review emphasizes the need for interdisciplinary research and collaboration with stakeholders to translate fundamental research into practical agricultural solutions.

Most plant embryos can survive for years in a dry state of less than 10% water( 1 ). Rehydration during seed germination is critical but risky–too little environmental water can dehydrate and kill the developing embryo. Plants avoid this by germinating only when sufficient water is present. However, the mechanisms by which sufficient water is sensed and how it triggers germination remain unknown. FLOE1 suppresses seed germination under water limitation by undergoing reversible phase changes in response to water potential( 2 ). To understand how FLOE1 affects germination, we compared water behavior in wild type and floe1-1 null mutants. While both had similar amounts of water when dry, floe1-1 seeds hydrated less than the wild type after imbibition. Additionally, bound water was less restricted in floe1-1 embryos, suggesting FLOE1 promotes macromolecular water binding. By developing plant-compatible versions of Genetically Encoded Multimeric nanoparticles (GEMs)( 3 ), we show that FLOE1 increases cytoplasmic viscosity during embryo rehydration. FLOE1 in yeast was sufficient to increase cytoplasmic viscosity. FLOE1 also increased cytoplasmic viscosity during hyperosmotic stress in yeast, and this ability was ablated by FLOE1 domain deletions. Our study identifies FLOE1 as a regulator of water content, dynamics, and cytoplasmic viscosity, linking molecular water control to cellular physical properties. ### Competing Interest Statement The authors have declared no competing interest.

Nitrogen is an essential element in all organisms, and its availability and use efficiency directly impact organismal growth and performance, especially in plants. Aminotransferases are core enzymes of the nitrogen metabolic network for synthesizing various organonitrogen compounds. Although each aminotransferase can potentially catalyse hundreds of transamination reactions with different combinations of amino and keto acid substrates, the full functionality of many aminotransferases remains elusive. Here we employed high-throughput gene synthesis and enzyme assay platforms to determine the substrate specificities of 38 aminotransferases of Arabidopsis thaliana and unveiled many previously unrecognized activities among a total of 4,104 reactions tested. The integration of these biochemical data in an enzyme-constrained metabolic model of Arabidopsis and in silico simulation further revealed that the promiscuity of aminotransferases may alter nitrogen distribution profiles and contribute to the robustness of the nitrogen metabolic network. This study provides foundational knowledge for deciphering the plant nitrogen metabolic network and improving nitrogen use efficiency in crops. Systematic characterization of Arabidopsis aminotransferase family enzymes uncovered many previously unrecognized activities and revealed their multi-substrate specificity, aspects that probably contribute to the robustness of the nitrogen metabolic network.

The transition from vegetative to reproductive growth is vital for plant fitness and crop yield and is strongly influenced by nutrient availability. While nitrogen deficiency accelerates flowering, phosphorus (P) limitation delays it. However, the molecular basis for how P availability regulates flowering time remains unclear. Here, through genome-wide association mapping in Arabidopsis, we uncover genetic variation in β-GLUCOSIDASE 25 (bGLU25) that modulates flowering under P-limited conditions. In P-sufficient environments, bGLU25 localizes to the endoplasmic reticulum. Under P limitation, however, bGLU25 translocates to the cytosol, a process mediated by P-regulated SERINE CARBOXY PEPTIDASE 50 (SCP50). In the cytosol, bGLU25 binds to JACALIN-LECTIN LIKE1 (AtJAC1), preventing the nuclear translocation of the Flowering Locus C (FLC) regulator GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7). This cytosolic sequestration of GRP7 under P-deprivation elevates FLC expression, delaying flowering. Moreover, in the monocot rice, the homologs of bGLU25 also modulate flowering responses to P availability, indicating a conserved role for bGLU25 across flowering plants. Our findings provide a molecular framework for breeding strategies aimed at optimizing flowering time in response to P levels. ### Competing Interest Statement The authors have declared no competing interest.

The accelerated pace of climate change over the past several years should serve as a wake-up call for all scientists, farmers, and decision makers, as it severely threatens our food supply and could result in famine, migration, war, and an overall destabilization of our society. Rapid and significant changes are therefore needed in the way we conduct research on plant resilience, develop new crop varieties, and cultivate those crops in our agricultural systems. Here, we describe the main bottlenecks for these processes and outline a set of key recommendations on how to accelerate research in this critical area for our society.

Cell types are fundamental functional units that can be traced across the tree of life. Rapid advances in single-cell technologies, coupled with the phylogenetic expansion in genome sequencing, present opportunities for the molecular characterization of cells across a broad range of organisms. Despite these developments, our understanding of eukaryotic cell diversity remains limited and we are far from decoding this diversity from genome sequences. Here we introduce the Biodiversity Cell Atlas initiative, which aims to create comprehensive single-cell molecular atlases across the eukaryotic tree of life. This community effort will be phylogenetically informed, rely on high-quality genomes and use shared standards to facilitate comparisons across species. The Biodiversity Cell Atlas aspires to deepen our understanding of the evolution and diversity of life at the cellular level, encompassing gene regulatory programs, differentiation trajectories, cell-type-specific molecular profiles and inter-organismal interactions.

Animals are sensitive to selective pressures associated with nutrient acquisition, underscoring the evolutionary significance of chemosensation in foraging and its intersection with satiety. For the model nematode Caenorhabditis elegans, isoamyl alcohol (3-methyl-1-butanol) and 2-methyl-1-butanol are produced by microbial fermentation and present in bacterial food sources collected from the natural environments. Both compounds, which are structural isomers of one another, elicit strong attraction in laboratory settings. Using laboratory chemotaxis assays, we show that starvation attenuates attraction to both compounds. Well-fed C. elegans is largely insensitive to the biosynthetic precursors of both alcohols, with the exception of 4-methyl-2-oxovaleric acid, which is a mild repellent. C. elegans chemosensation relies on expression of tax-4 cyclic nucleotide-gated and osm-9 transient receptor potential, vanilloid (TRPV) ion channels. Animals lacking both tax-4 and osm-9 are taste- and smell-blind. Animals lacking tax-4 fail to attract isoamyl alcohol and 2-methyl-1-butanol and those lacking osm-9 exhibit stronger attraction than the wild type. Starvation not only attenuates attraction, but also enhances repulsion to 4-methyl-2-oxovaleric acid and uncovers repulsion in tax-4 mutants absent in their well-fed counterparts. Collectively, these findings implicate satiety in regulating response strength, tax-4-dependent chemotaxis in attraction to isoamyl alcohol and 2-methyl-1-butanol, and osm-9-dependent chemotaxis in suppressing responses to biosynthetic precursors.

For most species, transcriptome data are much more readily available than genome data. Without a reference genome, gene calling is cumbersome and inaccurate because of the high degree of redundancy in de novo transcriptome assemblies. To simplify and increase the accuracy of de novo transcriptome assembly in the absence of a reference genome, we developed UnigeneFinder. Combining several clustering methods, UnigeneFinder substantially reduces the redundancy typical of raw transcriptome assemblies. This pipeline offers an effective solution to the problem of inflated transcript numbers, achieving a closer representation of the actual underlying genome. UnigeneFinder performs comparably or better, compared with existing tools, on plant species with varying genome complexities. UnigeneFinder is the only available transcriptome redundancy solution that fully automates the generation of primary transcript, coding region, and protein sequences, analogous to those available for high-quality reference genomes. These features, coupled with the pipeline's cross-platform implementation, focus on automation, and an accessible, user-friendly interface, make UnigeneFinder a useful tool for many downstream sequence-based analyses in nonmodel organisms lacking a reference genome, including differential gene expression analysis, accurate ortholog identification, functional enrichments, and evolutionary analyses. UnigeneFinder also runs efficiently both on high-performance computing (HPC) systems and personal computers, further reducing barriers to use.

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction, and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multi-well plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals, but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

Physiologically relevant drought stress is difficult to apply consistently, and the heterogeneity in experimental design, growth conditions, and sampling schemes make it challenging to compare water deficit studies in plants. Here, we re-analyzed hundreds of drought gene expression experiments across diverse model and crop species and quantified the variability across studies. We found that drought studies are surprisingly uncomparable, even when accounting for differences in genotype, environment, drought severity, and method of drying. Many studies, including most Arabidopsis work, lack high-quality phenotypic and physiological datasets to accompany gene expression, making it impossible to assess the severity or in some cases the occurrence of water deficit stress events. From these datasets, we developed supervised learning classifiers that can accurately predict if RNA-seq samples have experienced a physiologically relevant drought stress, and suggest this can be used as a quality control for future studies. Together, our analyses highlight the need for more community standardization, and the importance of paired physiology data to quantify stress severity for reproducibility and future data analyses.

The Plant Metabolic Network (PMN) is a free online database of plant metabolism available at https://plantcyc.org. The latest release, PMN 16, provides metabolic databases representing > 1200 metabolic pathways, 1.3 million enzymes, > 8000 metabolites, > 10 000 reactions and > 15 000 citations for 155 plant and green algal genomes, as well as a pan-plant reference database called PlantCyc. This release contains 29 additional genomes compared with PMN 15, including species listed by the African Orphan Crop Consortium and nonflowering plant species. Furthermore, 52 new enzymes with experimentally supported function information have been included in this release. The single-species databases contain a combination of experimental information from the literature and computationally predicted information obtained through PMN's database generation pipeline for a single species, while PlantCyc contains only experimental information but for any species within Viridiplantae. PMN is a comprehensive resource for querying , visualizing , analyzing and interpreting omics data with metabolic knowledge. It also serves as a useful and interactive tool for teaching plant metabolism. [GRAPHICS]

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multiwell plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

Noninvasive phenotyping can quantify dynamic plant growth processes at higher temporal resolution than destructive phenotyping and can reveal phenomena that would be missed by end-point analysis alone. Additionally, whole-plant phenotyping can identify growth conditions that are optimal for both above- and below-ground tissues. However, noninvasive, whole-plant phenotyping approaches available today are generally expensive, complex, and non-modular. We developed a low-cost and versatile approach to noninvasively measure whole-plant physiology over time by growing plants in isolated hydroponic chambers. We demonstrate the versatility of our approach by measuring whole-plant biomass accumulation, water use, and water use efficiency every two days on unstressed and osmotically stressed sorghum accessions. We identified relationships between root zone acidification and photosynthesis on whole-plant water use efficiency over time. Our system can be implemented using cheap, basic components, requires no specific technical expertise, and should be suitable for any non-aquatic vascular plant species.

Rhamnose is an essential component of the plant cell wall and is synthesized from uridine diphosphate (UDP)-glucose by the RHAMNOSE1 (RHM1) enzyme. RHM1 localizes to biomolecular condensates in plants, but their identity, formation, and function remain elusive. Combining live imaging, genetics, and biochemical approaches in Arabidopsis and heterologous systems, we show that RHM1 alone is sufficient to form enzymatically active condensates, which we name rhamnosomes. Rhamnosome formation is required for UDP-rhamnose synthesis and organ development. Overall, our study demonstrates a novel role for biomolecular condensation in metabolism and organismal development, and provides further support for how organisms have harnessed this biophysical process to regulate small molecule metabolism.

Climate change has already caused noticeable changes in species-wide traits, such as the well-documented acceleration of spring flowering. Because the evolutionary past has favored certain combinations of traits, some strategies like fast growth with early flowering that are adaptive today are at odds with other plant resilience strategies such as elevated water use efficiency. We know that the evolution of trait combinations is shaped by genomic constraints, but it is unclear whether and how this is affected by natural selection from climate change. Growing hundreds of Arabidopsis thaliana natural populations under different rainfall regimes revealed opposing natural selection on flowering time and water use efficiency, with strong antagonistic genetic correlations and contrasting causal alleles identified by Genome-Wide Association analyses. Inactivation of the central flowering regulator FLC in multiple, diverse accessions relaxed trait correlations in a genetic background-dependent manner and allowed for the emergence of a novel adaptive trait combination—early flowering and intermediate water use efficiency. Future climates are predicted to escalate conflicts in natural selection among adaptive traits, but our work shows that surprisingly simple genetic changes can help solve these conflicts.

Nutrient sensing and signaling are essential for adjusting growth and development to available resources. Deprivation of the essential mineral phosphorus (P) inhibits root growth.1 The molecular processes that sense P limitation to trigger early root growth inhibition are not known yet. Target of rapamycin (TOR) kinase is a central regulatory hub in eukaryotes to adapt growth to internal and external nutritional cues.2,3 How nutritional signals are transduced to TOR to control plant growth remains unclear. Here, we identify Arabidopsis-root-specific kinase 1 (ARSK1), which attenuates initial root growth inhibition in response to P limitation. We demonstrate that ARSK1 phosphorylates and stabilizes the regulatory-associated protein of TOR 1B (RAPTOR1B), a component of the TOR complex 1, to adjust root growth to P availability. These findings uncover signaling components acting upstream of TOR to balance growth to P availability.

This review highlights recent literature on biomolecular condensates in plant development and discusses challenges for fully dissecting their functional roles. Plant developmental biology has been inundated with descriptive examples of biomolecular condensate formation, but it is only recently that mechanistic understanding has been forthcoming. Here, we discuss recent examples of potential roles biomolecular condensates play at different stages of the plant life cycle. We group these examples based on putative molecular functions, including sequestering interacting components, enhancing dwell time, and interacting with cytoplasmic biophysical properties in response to environmental change. We explore how these mechanisms could modulate plant development in response to environmental inputs and discuss challenges and opportunities for further research into deciphering molecular mechanisms to better understand the diverse roles that biomolecular condensates exert on life.