The Plant Cell

T-DNA insertion mutants are widely used to disrupt genes and infer their functions, yet the insertions can also trigger unintended genomic changes that confound phenotypic interpretation. Here, we used T-DNA insertion mutants affecting the major mitochondrial malate dehydrogenase (MDH1) and the heterodimeric NAD-dependent malic enzymes (ME1 and ME2) to examine their functional coordination across photoperiods and irradiance regimes. Under short days, especially at low light intensity, mdh1xme2 mutants were markedly smaller than wild type and, unexpectedly, than the mdh1xme1xme2 triple mutant, and they showed a more pronounced reduction in photosynthetic capacity. ME1 was undetectable in mdh1xme2, implying that the double and triple mutants effectively lack heterodimeric ME and should therefore behave similarly, contrary to what we observed. Whole-genome analysis resolved this discrepancy by revealing that the MDH1 T-DNA insertion in mdh1xme2 is accompanied by a major rearrangement, a 137-kbp duplication downstream of the insertion site, which was absent in the mdh1xme1xme2 triple mutant. This duplication increased gene dosage and elevated transcript abundance across the duplicated interval, while proteomics detected 5 of the 38 encoded proteins, including PEPC1. mdh1xme2 accumulated oxaloacetate-derived amino acids and displayed an altered carbon/nitrogen balance, making PEPC1 a plausible contributor to the exacerbated mdh1xme2 phenotype. Together, our data indicate that a T-DNA-linked structural variant can amplify expression of dozens of genes and intensify phenotypes at specific conditions, thereby affecting the interpretation of genotype-phenotype relationships. Because Agrobacterium-mediated DNA transfer also underpins many genome-editing workflows, our findings argue that structural validation around insertion/editing loci should be considered essential when interpreting T-DNA-derived plant lines.

Plant survival and growth depend partly on the ability to manage energy resources in response to changing environmental conditions. SnRK1 plays a central role in this process by restricting growth under energy-limiting conditions while promoting stress adaptation and survival. When activated, SnRK1 triggers transcriptional reprogramming that prioritizes energy-producing pathways. A key mediator of this response is the transcription factor bZIP63, whose activity is regulated by SnRK1-dependent phosphorylation. Given its roles in energy homeostasis and its interaction with the circadian clock, bZIP63 influences growth and is therefore a candidate component of the Metabolic Daylength Measurement (MDLM) system, which integrates starch and sucrose metabolism with circadian timing and photosynthetic duration to regulate vegetative growth under contrasting photoperiods. We show that 39 bZIP63 direct targets regulated by SnRK1 correspond to a subset of short-day-induced genes associated with the MDLM system and are downregulated in a bZIP63 T-DNA mutant (bzip63-2) and/or in an RNAi-induced silencing line (RNAiWs_L9). Downregulation of these genes was more extensive in RNAiWs_L9 than in bzip63-2, possibly due to the unexplained silencing of BAM4, a {beta}-amylase that promotes starch degradation. Under short-day conditions, the frameshift mutant bzip63-5 (Col-0), bzip63-2 (Ws), and the bzip1-1/bzip53-1/bzip63-5 (Col-0) triple mutant, which disrupts bZIP63 heterodimerization partners, showed similar deregulation of a subset of these genes and comparable growth inhibition, whereas both growth and gene deregulation were more strongly affected in RNAiWs_L9. We further show in two partially complemented bzip63-2 lines that bZIP63 protein levels increase toward the end of the night and decline toward the end of the day, in synchrony with the diel oscillation of its transcript. Additional analyses of these lines, together with bzip63-2 line overexpressing bZIP63, suggest that the timing and amplitude of bZIP63 accumulation contribute to shaping the expression profiles of a subset of the 39 MDLM-associated genes. Together, these findings indicate that bZIP63 participates in a regulatory network linking SnRK1 signaling, photoperiod-changes, and growth within the MDLM system.

In flowering plants, pollen tubes communicate with ovular cells to achieve precise one-to-one pollen tube reception. The final step of this communication between the pollen tube and synergid cells has been extensively investigated and visualized by calcium imaging. Synergid cells exhibit characteristic cytoplasmic calcium concentration oscillations, which are thought to play a critical role in pollen tube reception. However, their significance and relationship with calcium dynamics in the entire ovule remain unclear. Here, we show, using the calcium sensor GCaMP6s, that proteins involved in asparagine-linked glycosylation (N-linked glycosylation) are required for normal calcium oscillations in synergid cells but are not essential for pollen tube reception. Using a semi-in vivo assay in Arabidopsis thaliana, we found that the amplitude of these oscillations prior to rapid pollen tube growth across the filiform apparatus was reduced in mutants lacking the oligosaccharyltransferase (OST) 3/6 subunit or alpha1,2-glucosyltransferase (ALG) 10, both of which are involved in N-linked glycosylation. Notably, these mutants did not exhibit reduced fertility attributable to defects in the female gametophyte but instead showed a polytubey phenotype due to a sporophytic defect. These findings suggest that N-linked glycans mediate communication between synergid cells and the pollen tube and indicate that the typical pattern of calcium oscillations in synergid cells is not essential for triggering pollen tube rupture. Furthermore, we show that sporophytic tissues of the ovule exhibit calcium waves that propagate toward the funiculus in correlation with pollen tube contact and rupture, implying that ovular tissues can potentially transmit these signals distantly beyond the ovule. Together, these findings reveal previously unrecognized intercellular calcium signaling and its significance in pollen tube reception by the ovule.

Chloroplast HSP70 is an essential component of the plastid proteostasis network, supporting protein folding, complex assembly and disassembly, and stress acclimation. Despite extensive genetic evidence for its essentiality, the cellular consequences of reduced chloroplast HSP70 activity remain poorly defined. Here, we investigated the function of the sole chloroplast HSP70 in Chlamydomonas reinhardtii, HSP70B, using an inducible artificial microRNA approach that reduced HSP70B abundance to below 30% of wild-type levels. HSP70B depletion resulted in cell division arrest and extensive proteome remodeling, characterized by strong upregulation of proteins involved in chloroplast protein quality control and membrane remodeling. Notably, this response was accompanied by increased abundance of protein quality control components in the endoplasmic reticulum, cytosol, and mitochondria, indicating pronounced proteostasis cross-talk between cellular compartments. In contrast, chloroplast and cytosolic ribosomes, photosynthetic and respiratory protein complexes, and central metabolic enzymes were broadly depleted, consistent with a collapse of cellular proteostasis. At the ultrastructural level, HSP70B-depleted cells exhibited lesions at thylakoid membrane conversion zones previously described in VIPP1-depleted cells. Accordingly, higher-order oligomeric forms of VIPP1 accumulated, and cells displayed extreme sensitivity to high-light stress. These findings confirm HSP70B as a key regulator of VIPP1 oligomer dynamics and highlight its central role in coordinating chloroplast membrane remodeling with cellular proteostasis in Chlamydomonas. One-sentence summaryDepletion of chloroplast HSP70B causes cell division arrest, proteostasis collapse, impaired VIPP1 oligomer dynamics with aberrant thylakoid structures, and increased light sensitivity.

Soil salinization is a growing global threat that limits crop productivity. To cope with sodium (Na) stress, plants have evolved tolerance mechanisms, including excluding Na from shoot tissues and tolerating elevated Na within shoots through tissue- and cellular-level mechanisms. Most current knowledge of Na accumulation comes from organ- or whole-plant measurements that lack the spatial resolution needed to resolve cellular tolerance mechanisms. Here, we used histological approaches to map leaf Na distribution in tomato (Solanum) species with contrasting salt-tolerance strategies. In the Na-excluding domesticated tomato (cv. M82), Na was largely confined to the bundle sheath, whereas Na-including wild relatives accumulated Na throughout the blade mesophyll. Consistent with these cell population-specific Na patterns, M82, but not S. pennellii, exhibited reduced symplastic transport and plasmodesmal permeability under salt stress. A genetic screen combined with transcriptome profiling implicated Plasmodesmata-Located Protein 1 (PDLP1), a regulator of callose-mediated plasmodesmal closure, in establishing symplastic domains in M82 that restrict Na movement into the mesophyll. Moreover, PDLP1 expression negatively correlated with mesophyll Na+ levels across wild and domesticated tomatoes. Collectively, these results link cellular Na enrichment patterns to symplastic connectivity and suggest that PDLP1-mediated regulation of plasmodesmata contributes to leaf-level salt-tolerance strategies. HighlightsO_LICell type-specific Na accumulation differs between domesticated tomato (Solanum lycopersicum cv. M82) and its wild relative S. pennellii. C_LIO_LIAdditional salt-tolerant wild tomato relatives exhibit leaf Na enrichment patterns similar to S. pennellii. C_LIO_LISalt stress reduces symplastic transport and plasmodesmal permeability in M82 leaves but not in S. pennellii. C_LIO_LIAn introgression line (IL6-4) between the two tomato species, which carries S. pennellii Plasmodesmata-Located Protein 1 (SpPDLP1), shows S. pennellii-like Na enrichment patterns. C_LIO_LIPDLP1 expression shows a negative correlation with mesophyll Na+ levels across tomato species. C_LI

Understanding why sex chromosomes repeatedly evolve recombination suppression, gene loss, and repeat accumulation remains a central challenge in evolutionary genomics. Plant sex chromosomes may be particularly informative, because they have often evolved recently from hermaphroditic ancestors. We studied the sex-linked region of the dioecious annual Mercurialis annua using new long-read genome assemblies of an XX female and a YY male, a published female assembly, linkage maps, and population-genomic data from several Mercurialis species. We identify two discrete nested evolutionary strata on the Y chromosome of diploid M. annua. A young stratum was generated by a large inversion and shows little degeneration, whereas an older stratum nested within it exhibits substantial gene loss, transposable-element accumulation, insertion of paralogous gene copies, and elevated X-Y sequence divergence. These findings indicate that recombination suppression evolved in at least two stages, with a recent inversion expanding an older non-recombining region. Comparative analyses among several Mercurialis species further show that the extent of sex-linked differentiation varies markedly among them. We also identify APRR7 as the only gene showing consistent male-specific inheritance across the genus; this gene is a strong candidate master sex-determination gene. Together, our results refine the structure and gene content of the sex-linked region in M. annua and contribute to our understanding of the diversity of sex chromosomes in plants.

Metal homeostasis in plants relies on coordinated uptake, chelation, and transport mechanisms involving, but not limited to, citrate and nicotianamine (NA). In Arabidopsis (Arabidopsis thaliana), disruption of the citrate exporter FRD3 (FERRIC REDUCTASE DEFECTIVE 3) causes constitutive Fe deficiency responses, altered iron (Fe), manganese (Mn) and zinc (Zn) distribution, with Fe accumulation in the root cell wall. This ultimately results in oxidative and biotic stress responses, and impaired root development, phenotypes that are partially alleviated by Zn excess. In this study, we investigated the consequences of impairing both citrate loading into xylem vessels and NA partitioning within cells. The frd3 zif1 double mutant exhibits enhanced sensitivity to Zn excess, severe defects in root system architecture and meristem maintenance, persistent oxidative stress, and compromised reproductive development. These phenotypes correlate with sustained activation of Fe deficiency signaling and marked defects in root-to-shoot metal translocation. Our findings reveal that coordinated citrate export and NA compartmentation form an integrated buffering strategy required to maintain metal homeostasis and partitioning, as well as redox balance and proper development, including root plasticity and seed yield, under fluctuating metal availability.

Hundreds of proteins in the cell require iron (Fe) or Fe-containing cofactors to function. However, how Fe2+ or Fe3+ are specifically allocated to each of these proteins in plant cells remains largely unknown. It has been proposed that Fe metalation could be driven by specific interactions with Fe-shuttling proteins known as Fe-chaperones. Here, we present the first family of plant Fe2+-chaperones (ICHAPs) with orthologues in dicots and monocots. The role of these proteins in Fe distribution to Fe-dependent metabolic processes has been illustrated using symbiotic nitrogen fixation in Medicago truncatula root nodules. ICHAP1 is a soluble Fe2+-binding protein that interacts with plasma membrane Fe2+ transporter NRAMP1, but not with symbiosome Fe2+-transporters. ICHAP1 mutants present altered Fe distribution in cells and they cannot fix nitrogen. A second family member, ICHAP2 is required to target Fe2+ to symbiosomes, as it accepts Fe2+ from ICHAP1 and interacts with symbiosome Fe2+-importer VTL8, but not with NRAMP1. These results indicate a path for Fe2+ allocation from the plasma membrane to the symbiosome through specific protein-protein interactions and Fe2+ exchange from NRAMP1 to ICHAP1, to ICHAP2, and to VTL8.

In C4 photosynthesis, incoming CO2 is incorporated in mesophyll cells (MC) into 4-carbon acids that diffuse to bundle sheath cells (BSC) and decarboxylated to generate a high CO2 concentration that suppresses the oxygenation reaction of Rubisco. Decarboxylation can occur by NADP-malic enzyme, (NADP-ME), NAD-malic enzyme (NAD-ME) or phosphoenolpyruvate carboxykinase (PEPCK). NADP-ME generates NADPH in the BSC chloroplast and species that use it as the major route for decarboxylation typically have dimorphic BSC chloroplasts with little or no photosystem II. They operate an energy shuttle: much of the 3-phosphoglycerate formed in the Calvin-Benson cycle diffuses to the MC, enters the chloroplasts and is reduced to triose phosphates that return to the BSC. In species where carboxylation occurs mainly via NAD-ME or PEPCK, BSC chloroplasts possess photosystem II. Indirect evidence indicates they nevertheless have the capacity to operate an energy shuttle. We show here that NAD-ME and PEPCK species possess large pools of 3PGA and triose phosphates and, for two examples of each subtype, opposed concentration gradients of 3-phosphoglycerate and triose phosphates to drive rapid exchange between the BSC and MC. Reasons for and consequences of the widespread operation of the intercellular energy shuttle in C4 plants are discussed. Highlight StatementAn intercellular energy shuttle in which 3-phosphoglycerate moves from the bundle sheath to the mesophyll and triose phosphates return to the bundle sheath is a general feature of C4 photosynthesis.

Durable resistance to soil-borne pathogens remains elusive in cereals, partly because susceptibility (S) genes that facilitate root infection have not been identified in monocots. In the model legume Medicago truncatula, the SCAR/WAVE complex member MtAPI functions as a root S-gene for microbial invasion. Whether SCAR gene associated susceptibility function is conserved in monocots, and whether SCAR gene inactivation can enhance root resistance in cereals, remains unknown. Here, we identify and characterize three SCAR genes in barley: HvSCAR-A, HvSCAR-B, and HvSCAR-C. Cross-species complementation assays indicate that HvSCAR-B and HvSCAR-C are functionally similar to MtAPI. While hscar-b and hvscar-c single mutants exhibited no major growth defects, hvscar-a mutants showed strongly reduced seed production, and a hvscar-b/c double mutant displayed shorter root hairs. Notably, the hvscar-b/c double mutant exhibited increased resistance to the hemibiotrophic pathogen Phytophthora palmivora but greater colonization by the symbiotic arbuscular mycorrhizal fungus Funneliformis mosseae, underscoring a complex role in plant root - microbe interactions. Our findings reveal a conserved susceptibility function of SCAR genes in monocots and identify api monocot homologs as promising targets for engineering disease resistance in cereals. This study offers new insights into SCAR protein functional diversification and its potential for improving root health in crop plants.

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

Computational tools, including biological language models (LMs), show substantial promise in predicting the impact of genetic variants on plant fitness. However, validating variant effect predictions (VEP) requires experimental populations where genetic variation consists of discrete point mutations rather than segregating recombination blocks. In this study, we generated a novel population of Brachypodium distachyon mutant lines to evaluate the accuracy of VEP at single-base resolution. These lines were advanced through single-seed descent for five generations (M1 to M5), with whole-genome sequencing performed at M2 and M5 and phenotypic measurements recorded at M3 and M4. Using state-of-the-art VEP models, we predicted the functional impact of missense protein-coding variants and gene-proximal non-coding variants. We validated these predictions by estimating the effect of mutations on whole-plant measurements (burden tests) and their probability of fixation from M2 to M5 (purging tests). Among missense variants, the protein LM ESM showed superior predictive accuracy compared to the bioinformatic standard SIFT and the genomic LM PlantCAD. Notably, the relationship between VEP scores and allele fixation suggested a log-linear relationship between VEP scores and variant fitness. Among gene-proximal variants, PlantCAD appeared more accurate than supervised models of regulatory activity, such as chromatin accessibility (a2z) and RNA abundance (PhytoExpr). Collectively, our findings highlight the utility of state-of-the-art VEP tools as predictors of fitness and demonstrate the potential of mutant populations to evaluate computational tools for precision breeding applications.

Fast-neutron mutagenesis creates diverse genome-wide mutations, providing a powerful tool for crop functional genomics. Here, we present an expanded genomic and phenotypic analysis of 3,268 fast-neutron (FN)-induced mutant rice lines (Oryza sativa L. cv. Kitaake). All FN lines were whole-genome sequenced, and mutations were identified by alignment in the Nipponbare and KitaakeX reference genomes. We cataloged over 428,000 mutations affecting 78.49% of Nipponbare genes and 70.38% of KitaakeX genes. In silico expression analysis indicates that 575 non-mutated Nipponbare genes are highly expressed and likely essential for viability. Each mutant carries, on average, 68.5 mutations in the Nipponbare alignments or 63.2 mutations for KitaakeX alignments, distributed randomly across all 12 chromosomes with no evident hotspots. FN lines have approximately 8.5% fewer mutations when using the KitaakeX alignment, underscoring the unique contributions of each reference genome and the importance of utilizing both for comprehensive mutation discovery. The majority of mutations are small deletions and single-base substitutions, with deletions predominating in their effect on genes. We found that 74.4% of all transcription factor Nipponbare genes were mutated at least once. Phenotypic characterization of over 2,700 lines revealed a broad spectrum of variation in core agronomic traits (heading date, tiller number, plant height, panicle weight, seed yield components) and other morphological variants of interest. The integration of genomic and phenotypic data through the KitBase platform enabled the identification of candidate genes for several traits of interest. The KitBase website (https://kitbase.ucdavis.edu) has been updated to provide open access to all mutation data and seed stocks, as well as an intuitive query interface, facilitating forward and reverse genetic analyses in rice. This expanded resource enriches the rice functional genomics toolkit and highlights the value of coupling high-density mutation mapping with phenotypic data for rapid gene discovery and crop improvement.

Macroautophagy is a conserved intracellular catabolic process in eukaryotes that participates in chloroplast degradation, through the selective breakdown of chloroplast components. Selective autophagy of membrane-bound organelles typically requires receptors that bridge organelle membranes and pre-autophagosomal structures. Here we identify OEP24.1 as a new receptor in the selective chloroplast piecemeal autophagy, supporting the degradation of stromal proteins. We found that the {beta}-barrel protein OEP24.1 is located at the outer membrane of plastid envelopes and on bodies budding off plastids into the cytosol and containing stroma proteins. OEP24.1 interacts physically with ATG8 autophagy proteins in a UIM dependent manner. OEP24.1-GFP and RFP-ATG8 colocalize with in mobile autophagosome-like puncta in the cytosol and in autophagic bodies within the vacuole. Delivery of OEP24.1 to vacuole lumen is dependent on active autophagy. OEP24.1 controls carbon allocation at the whole plant level, carbon concentrations in flowering stems and xylem composition. These phenotypes can be explained by the role of OEP24.1 in metabolite diffusion across the chloroplast envelope, and by its involvement in the facilitation of chloroplast quality control through piecemeal autophagy.

Salt stress alters plant development, including the floral transition, but regulation of timing of flowering by salt is poorly understood at the molecular level. To identify genetic loci regulating the floral transition under high soil salinity, we performed a genome-wide association study (GWAS) in Arabidopsis thaliana and identified natural variation at the UGT74E1-UGT74E2-BT3 (UUB) locus that correlates with bolting time specifically in response to salt stress. Genetic analysis revealed BT3 as a novel repressor of the floral transition in control conditions. Similarly, the putative IBA glycosylases UGT74E1 & UGT74E2 delay the floral transition in control conditions. Furthermore, we identified that IBA homeostasis regulators TOB1 and ECH2/IBR10 play a key role in the floral transition, and that ECH2/IBR10 are required for the early flowering phenotype of the ugt74e1/ugt74e2 double mutant, indicating that UGT74E1 & UGT74E2 delay flowering by altering IBA homeostasis. A pangenome analysis of the UUB locus revealed variation in the occurrence of the DNA transposon SAUERKRAUT (SKRT). CRISPR-mediated SKRT deletion in Col-0 affected gene expression both within and outside the UUB locus and caused a salt-dependent delayed floral transition. The delayed bolting phenotype of the skrt-2 mutant also depends on ECH2/IBR10 function, indicating that SKRT accelerates the floral transition by altering IBA homeostasis. Finally, targeted demethylation of SKRT resulted in delayed floral transition under salt stress. Taken together, our data show a role for SKRT and its DNA methylation levels in the salt-dependent bolting time response in Arabidopsis, revealing a novel molecular mechanism to control flowering in adverse conditions.

Alternative splicing (AS) is traditionally understood to increase transcript and protein diversity by generating multiple coding isoforms from a single gene. In addition, many alternative isoforms are degraded through non-sense mediated decay (NMD). Furthermore, we show here that AS also produces nuclear long non-coding RNAs (lncRNAs) from protein-coding genes, which can serve regulatory functions rather than expanding proteomic complexity. Focusing on the Arabidopsis thaliana SR protein gene At-RS31, we found that a non-coding isoform (mRNA3) accumulates in the nucleus under dark conditions. This transcript binds the protein product of its own transcriptional unit, modulating splicing decisions and balancing gene activity in response to environmental cues. Overexpression of mRNA3 down-regulates the action of At-RS31 on its target genes in trans and restores the phenotype induced by intron-less At-RS31 accumulation. Further sub-cellular fractionation and RT-PCR analyses show that many SR genes generate nuclear-retained non-coding isoforms, especially under dark conditions, suggesting a widespread mechanism. These findings redefine the role of AS in plants, highlighting its capacity to generate regulatory lncRNAs to fine-tune gene expression beyond protein diversification. Highlights- Alternative splicing generates regulatory lncRNAs from protein-coding genes - A nuclear-retained isoform forms an autoregulatory RNA-protein feedback loop - Non-coding isoforms of SR genes accumulate in the nucleus under dark conditions - Co-expression of the non-coding isoform rescues splicing factor overexpression phenotypes

Terrestrial plants emerged from the water about 500 million years ago. Thereafter, they have diversified and now inhabit most of the Earths surface. More recently, some species have re-adapted to an aquatic lifestyle, both in fresh and salt water, and fully or partially submerged. The mechanisms enabling these adaptations between terrestrial and aquatic life are extremely numerous, making it difficult to have a comprehensive overview of the phenomenon. Here, we performed a series of intraspecific measurements of the selection pressure affecting orthologous genes in eight aquatic and four terrestrial plants. Our analyses showed that aquatic plants have a relaxed selection pressure on nutrient assimilation mechanisms, probably linked to a greater bioavailability, as well as stronger adaptations to oxidative stress, while terrestrial plants evolution is linked to environment perception. Inter-species analyses have also highlighted a different evolution of chloroplast proteins between these two types of plants, suggesting adaptations to gas availability.

Inositol phosphates (InsPs) and inositol pyrophosphates (PP-InsPs) are conserved signalling molecules, but their evolutionary origin and diversification in the green lineage remain poorly understood. Here we investigated the InsP network in the streptophyte alga Chara braunii, a key lineage lose to the origin of land plants. Using capillary electrophoresis-electrospray ionization mass spectrometry, we detected a broad spectrum of InsP and PP-InsP species from InsP3 to InsP8, including multiple positional isomers. Developmental profiling across dormant oospores, young thalli and mature thalli revealed extensive metabolic remodeling, with InsP6 as the dominant metabolite and distinct stage-dependent changes in lower InsPs and pyrophosphorylated species. Multiple PP-InsP5 and (PP)2-InsP4 isomers were identified, together with an unassigned additional InsP8-like signal, indicating further pathway complexity. Bioinformatic analyses identified candidate homologs of major InsP metabolic enzymes, supporting the presence of an enzymatic framework for InsP synthesis and turnover similar to land plants. Environmental perturbation revealed isomer-selective effects: prolonged light and dark phases strongly affected the accumulation of specific InsP5 and PP-InsP5 isomers, with 1-PP-InsP5 emerging as the most stimulus-responsive pyrophosphate species, whereas heat stress preferentially reduced 4-PP-InsP5. Together, these findings show that a structurally complex and environmentally responsive InsP network was already established in streptophyte algae before the emergence of land plants.

Self-incompatibility (SI), a reproductive mechanism that prevents self-pollen from fertilizing the ovule, is widespread in flowering plants, including the Brassicaceae family, where it promotes outcrossing, genetic diversity, and hybrid vigor. Although prevalent in Brassica rapa, an economically vital crop, it remains poorly characterized in widely grown varieties, such as toria and yellow sarson, with prior studies primarily focused on Brassica napus. Given its potential for hybrid breeding and crop improvement in rapeseed (B. rapa), we characterized key SI-regulatory genes, analyzing their phylogenetic relationships, structure-function dynamics, and expression patterns. Our results indicate sequence, structural, and functional homology as well as conservation with previously known candidates. This study identifies SRK, FER, and ARC1 as essential, while MLPK plays a minor role in SI for the varieties under study. Furthermore, we identified that SRK, FER, and MLPK activate ROS during the SI response, while ARC1 does not. Our findings establish a foundation for harnessing this natural system to integrate agriculturally important traits and sustain them across generations via outcrossing.

Mitochondrial genomes retain only a tiny number of genes from their bacterial progenitors, including key components of protein translation machinery. The set of mitochondrially encoded tRNAs and ribosomal subunits is highly variable across angiosperms, with many examples of mitochondrial gene loss, replacement, and/or transfer to the nucleus. This dynamic history suggests large-scale remodeling of mitochondrial translation machinery in some lineages, but such conclusions are largely inferred from genomic sequence and protein targeting predictions. Here, we use proteomic (LC-MS/MS) analysis of purified mitochondria and chloroplasts from angiosperm species with major differences in mitochondrial gene content (Arabidopsis thaliana and Silene conica). Our analysis largely confirms the current understanding of subcellular localization for nuclear-encoded proteins involved in tRNA metabolism and ribosome function in A. thaliana, although some aminoacyl-tRNA synthetases (aaRSs) may have more specialized subcellular roles than previously thought. In contrast, S. conica has undergone extensive mitochondrial gene loss and numerous associated changes in the composition of its mitochondrial proteome, including apparent retargeting of aaRSs, replacement of ribosomal subunits, and loss of the glutamine amidotransferase (GatCAB) complex. Overall, this analysis illustrates how the complex network of molecular interactions necessary for mitochondrial translation are perturbed by gene loss, transfer, and replacement.