The Plant Journal

Fitness costs of plant disease defence are often subtle and difficult to quantify. In this study, we therefore used comparative high-throughput phenotyping in two independent facilities to assess growth, morphology and physiology of potato (cv. Desiree) with high time-resolution monitoring different defence mechanisms under pathogen-free conditions. Plants were either treated weekly with the resistance inducers {beta}-aminobutyric acid (BABA; 10 mM) or potassium phosphite (KPhi; 36 mM) or comprised six transgenic lines expressing late blight resistance genes (single Rpi genes or a three-gene stack) or reduced jasmonate perception (StCOI1-RNAi). Over four weeks, image-derived traits revealed consistent cross-facility effects for plant height and colour: BABA treatment increased plant height but reduced canopy area and induced a paler greenness signature, whereas KPhi caused minimal and transient growth effects. Chlorophyll fluorescence at the NaPPI facility indicated reduced vitality (Rfd_Lss) in BABA-treated plants and increased Rfd_Lss following KPhi, while maximum PSII efficiency was largely unchanged. Several transgenic lines showed somewhat reduced above-ground biomass. Enzyme activity profiling produced distinct treatment and genotype signatures, but was strongly modulated by facility conditions that overrode these specificities. Overall, high-throughput phenotyping robustly detected subtle growth-defence trade-offs across platforms. HighlightHigh-throughput optical phenotyping validated across two independent research facilities reveals that stacked resistance genes and resistance inducers in potato trigger subtle growth trade-offs. Graphical abstracts O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/713143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@89df47org.highwire.dtl.DTLVardef@1a1ce64org.highwire.dtl.DTLVardef@1f52f0dorg.highwire.dtl.DTLVardef@1e41c35_HPS_FORMAT_FIGEXP M_FIG C_FIG Experimental timeline for high-throughput plant phenotyping platforms. Created in BioRender. Poque, S. (2026) https://BioRender.com/nmkve7g

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA-protein interactions are commonly investigated using chromatin immunoprecipitation (ChIP), which relies on specific antibodies often involving technically demanding protocols. CRISPR-Cas technologies have enabled sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation to express the CRISPR machinery, limiting their applicability across species, tissues and physiological contexts. Here, we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation operating directly on purified plant nuclei. In GRASP, dCas9-gRNA ribonucleoprotein complexes are used to capture predefined genomic regions from chromatin under native conditions, bypassing the need for transgene expression. Using grapevine and tomato as model systems, we demonstrate efficient and highly specific enrichment of target loci, including telomeric repeats as well as low-copy and single-copy genomic regions, with qPCR and NGS validation. These results establish GRASP as a robust and broadly applicable platform for locus-specific chromatin isolation in plants. Beyond sequence-specific DNA isolation, GRASP establishes a versatile platform for potential downstream analyses of locus-associated chromatin components, including protein complexes, distal DNA-DNA interactions and chromatin-associated RNAs, providing new opportunities to investigate regulatory architecture in plant genomes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/712347v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@13758e8org.highwire.dtl.DTLVardef@adfd82org.highwire.dtl.DTLVardef@de81f4org.highwire.dtl.DTLVardef@25c2d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Long-read sequencing has shown a rapid technological development during the last years. It has been established as the standard method for the sequencing of plant genomes and has also gained importance for full plasmid sequencing. As Sanger sequencing has a limited read length of about 1 kb, long read sequencing offers a great advantage, as the full plasmid can be sequenced in one read. Here, we present a cost-effective workflow to sequence full plasmids and compare the results against an expectation. The per plasmid cost of this workflow is determined by the number of plasmids investigated simultaneously, but can be lower than the price of a single Sanger sequencing reaction. We developed a workflow for automatic data processing, which allows us to complete sequencing and data analysis within a day.

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.

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.

Malate and fumarate constitute a significant transient carbon stock that is dynamically synthesized during the photoperiod. These organic acids are diurnally stored and remobilised from the vacuole, and they have a key role in the cellular metabolic regulation. This function is well known in C4 and CAM plants. However, in C3 species that are the majority of terrestrial plants, the importance of the vacuolar accumulation/release and its influence on plant growth is still an open question. In Here we addressed this issue generating multiple knockout mutants in Arabidopsis thaliana lacking vacuolar anion channels of the Aluminium-Activated Malate Transporter (ALMT) family, to impair malate and fumarate transport to the vacuole. We show that in these mutants reducing vacuolar transport of malate and fumarate in mesophyll cells leads to a dramatic growth impairment. Metabolic and fluxomic analysis revealed that vacuolar malate and fumarate transport influences plant carbon and nitrogen metabolism as well as cellular pH and ionic homeostasis. In conclusion, our results show that the transport organic acids like malate and fumarate across the vacuolar membrane is essential for plant growth in a C3 plant too. These results establish the importance of the vacuolar pools of malate and fumarate in plant metabolism.

Common buckwheat (Fagopyrum esculentum Moench) is a globally cultivated pseudocereal with a high nutritional quality and economic value. Due to its self-incompatibility, common buckwheat exhibits a high level of heterozygosity, making genome assembly challenging. Consequently, reference-level haplotype-resolved assemblies of common buckwheat are scarce, hindering research and genomics-assisted breeding. Here, we present a near-complete, chromosome-level, haplotype-resolved assembly of a common buckwheat F1 genotype (named Tuka), generated using a trio-binning approach that integrated parental Illumina short-read data with PacBio HiFi and Hi-C data from Tuka. The Tuka assembly comprises two haplomes, Tuka_h1 and Tuka_h2, both showing high contiguity (contig N50 of 76.68 Mb and 84.57 Mb, respectively), high completeness (assembly sizes of 1.28 Gb and 1.23 Gb with BUSCO scores of 96.9% and 96.8%, respectively), high base-level accuracy (QV of 59.08 and 63.03, respectively), and few gaps (35 and 30, respectively). This near-complete assembly of Tuka serves as a valuable genomic resource for common buckwheat, enabling advanced genomic analyses and accelerating research and breeding using state-of-the-art genomic tools.

The combination of target capture sequencing (TCS) with low-coverage whole genome sequencing (lcWGS), an approach known as Hyb-Seq, has allowed the integration of natural history collections into the genomics revolution, transforming biodiversity research. To implement Hyb-Seq, a collection of genomic targets, often nuclear orthologs, is needed to design probes for TCS. In flowering plants, the universal Angiosperms353 probe set has been proven resolutive at multiple evolutionary scales, with caveats. Malpighiales is known to be one of the most challenging flowering plant orders to resolve. Within this order, the clusioid clade ([~]2.2K species, 94 genera, five families) is no exception. To resolve phylogenetic relationships in this recalcitrant clade, we design a custom probe set, the Clusioids626 kit, composed of 39,936 120-mer probes targeting 626 nuclear orthologs ([~]6.6M nucleotides). This probe set includes all Angiosperms353 targets and 273 clusioid-specific ones, carefully chosen taking copy-number, length evenness, and phylo-informativeness into account. We test our probe set on 70 accessions representing all families and tribes in the clusioid clade. On average, 50.4% of TCS reads mapped to our targets, recovering a median of [~]600 orthologs. Relationships for all clusioid families are fully resolved for our nuclear targets. Additionally, 105 plastid coding DNA sequences were retrieved from the lcWGS fraction. A strong cyto-nuclear conflict was detected. The Clusioids626 kit performs better than the universal Angiosperms353 enrichment panel alone. Our kit design workflow can be extended into other lineages for which a universal probe set exists but more resolution is needed.

DNA methylation plays a central role in the regulation of gene expression. In plants, methylation occurs in the CG, CHG and CHH contexts, via distinct DNA methyltransferases including MET1, CMT3 and the RNA-directed DNA Methylation (RdDM) pathway via DRM2. In interspecific hybrids, these epigenetic mechanisms are confronted to a mixed small RNA population and two subgenomes harbouring specific methylation patterns, therefore generating unique expression profiles. The aim of this work was to understand these regulations by analysing gene expression, DNA methylation and small RNAs in a Citrus hybrid resulting from the cross between C. reticulata (mandarin) and C. australasica (finger lime). Haplotype-resolved subgenomes assembly identified hundreds of allele-specifically expressed genes. Asymmetric reprogramming of methylation was observed, in particular an increase in CHH in C. australasica haplotype. Surprisingly, CHH methylation, usually associated with gene silencing, was correlated here with increased expression, but also 24nt small RNA populations at their promoter regions. Similar analyses of the parental lines and other citrus species suggest the correlation between CHH methylation-enriched promoter and high expression level is not due to the hybridization, but seem to be generally true for all citrus. These observations suggest that, in citrus fruit, RdDM could activate transcription. This work also provides a full pipeline to analyse the expression profiles and DNA methylation in complex hybrids, which could be crucial for anticipating varieties resistant to diseases and the current threats affecting citriculture such as the Huanglongbing disease.

The transition from hulled to free-threshing grain was a pivotal event in wheat domestication, enabling efficient harvesting and processing. Threshability in tetraploid wheat is controlled primarily by the Q locus and two Tenacious glume (Tg) loci on chromosomes 2A and 2B, yet the molecular basis of the major Tg1-B locus remains incompletely characterized. Here, we phenotyped a durum wheat x wild emmer wheat (WEW) recombinant inbred line (RIL) population across two field environments and performed QTL analysis for glume tenacity (TG), threshability ratio (THRR), and seed number per spike (SDNPS). A total of 19 significant QTLs were detected across six chromosomes. The largest-effect loci for both TG and THRR co-localized on chromosome 2B, with LOD scores up to 14.22 and phenotypic variance explained up to 31.2%, corresponding to the previously described Tg1-B locus. To validate this QTL, the donor RIL was backcrossed three times to Svevo to generate a near-isogenic line, NIL-65 (BC3F5), confirmed by whole-genome skim sequencing to carry a homozygous WEW introgression at Tg1-B. A segregating BC4F2 population derived from NIL-65 confirmed that plants homozygous for the dominant Tg1-B allele displayed significantly higher glume tenacity and intact glume morphology compared to tg1-B sister lines, which exhibited basal glume cracking characteristic of the free-threshing phenotype. Genotyping-by-sequencing delimited the causal interval to an approximately 11 Mb introgression on chromosome 2B. These results confirm the major role of Tg1-B in determining glume tenacity in tetraploid wheat, provide a validated near-isogenic germplasm resource, and lay the foundation for fine-mapping and functional characterization of the underlying gene(s).

Genomic imprinting is a rare epigenetic phenomenon in plants and animals, defined by parent-of-origin specific gene expression. Its molecular mechanisms and evolutionary significance remain incompletely understood. In this study, we investigated whether genomic imprinting occurs in Scots pine and, by extension, in other conifers to gain insight into the evolutionary origins of imprinting. We performed reciprocal crosses to assess imprinting in seed embryos and applied a unique approach that used exome-capture data from the haploid, maternally inherited megagametophyte tissue to identify maternal alleles, thereby allowing us to infer paternal alleles in the embryos of the same seeds. Our findings show that maternally inherited haploid megagametophyte tissue offers an effective strategy for resolving parental alleles in offspring while simultaneously removing extensive paralogous variation from the dataset. This framework is broadly applicable to other conifer species and to taxa that possess comparable maternally derived haploid tissues. No evidence of genomic imprinting was detected. Although the limited overlap between the exome-capture and RNA-sequencing datasets and the stringent paralog filtering reduced the amount of analyzable data considerably, the absence of detectable imprinting may also reflect genuinely weak or absent imprinting signals in conifers. We identified several limitations in this preliminary study and outline recommendations for future work to overcome them, and additional research will be necessary to determine whether genomic imprinting occurs in conifers

Drought is a major constraint on the productivity of durum wheat across Mediterranean and North African regions. To elucidate the mechanisms underlying drought resilience, we employed a combination of scenario-controlled phenomics and flag leaf transcriptomics across ten durum wheat genotypes. These included the Tunisian landraces Chili and Mahmoudi, seven breeding lines, and the reference cultivar Svevo. The plants were grown to maturity under well-watered or long-term drought conditions in pots and rhizotrons, enabling a comprehensive assessment of growth, yield components, root architecture, physiological traits, and reaction norm plasticity. Drought markedly reduced performance, yet Chili and Mahmoudi consistently maintained superior biomass, grain number and intrinsic water use efficiency (iWUE). This was supported by balanced C/N allocation, strong osmotic adjustment, and the ability to sustain robust root systems under stress, albeit through partly divergent physiological strategies. Transcriptomic profiling revealed highly genotype specific responses, with drought tolerance unrelated to the number of differentially expressed genes. Instead, the landraces displayed distinct regulatory programs involving mainly photosynthesis protection, ABA-related transporters, osmotic adjustment pathways, and stress-responsive transcription factors. These mechanistic insights identify actionable physiological and molecular determinants of drought plasticity and provide high value targets for accelerating the breeding of climate resilient durum wheat. HighlightsIntegrated phenomics and transcriptomics revealed landrace-specific physiological and molecular mechanisms enabling superior drought resilience and identifying actionable targets for durum wheat improvement.

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.

Small signaling peptides (SSPs) are critical regulators of plant growth, development, and responses to biotic and abiotic stress, yet their role in the C4 grass Sorghum bicolor is largely uncharacterized. To help fill this knowledge gap, 219 S. bicolor genes that encode SSPs were identified based on SSP sequences previously identified in Arabidopsis thaliana, Oryza sativa, Zea mays, Triticum aestivum, and Brachypodium distachyon. The 219 sorghum genes were assigned to 19 gene families, analyzed for the presence of motifs, and aligned with genes that encode SSPs in other plants using phylogenetic analysis. Expression of the 219 SSP encoding genes in sorghum organs, during stem development, and in stem tissues and cell types revealed distinct spatial, temporal and developmental patterns of expression. Genes associated with the SbCEP and SbRGF families were preferentially expressed in roots, whereas SbEPF genes were expressed in stems and panicles. The expression of genes during bioenergy sorghum stem growth and development was investigated because stems account for [~]80% of harvested biomass and serve as conduits for water and nutrient transport between leaves and roots. During stem development, 28 SSP encoding sorghum genes in several families (CLE, EPF, CEP, GASS, PSY, ES, PSK, CAPE, POE) were expressed at higher levels in zones of cell proliferation. For example, the TDIF homologs SbCLE41 and SbCLE42 were expressed at high levels in nascent stem nodes where they may regulate cambial activity and vascular bundle cell differentiation. A different set of 15 genes in the CIF, POE, CAPE, PSY, CEP, RALF, and CLE families were expressed at higher levels in zones of stem tissue differentiation highlighted by elevated expression of 5 SbRALFs in the stem nodal plexus. Cell type specific expression of many SSP encoding sorghum genes was also observed in fully elongated internodes indicating gene expression is regulated with high spatial resolution. Overall, the results provide a foundation of information for analysis of SSP functions in sorghum that can be integrated with knowledge of sorghum gene regulatory networks to modulate traits important for production of sorghum crops.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport (CET) around photosystem I (PSI) and contributes to photosynthetic regulation and photoprotection under various environmental stresses. Although NDH function has been extensively characterized under controlled conditions, NDH-deficient mutants often show only subtle phenotypes in such environments, leaving its physiological importance under naturally fluctuating field conditions poorly understood. Here, we evaluated growth, yield, and photosynthetic performance of NDH-deficient rice cultivated under field conditions. Mutant plants exhibited reduced biomass accumulation and grain yield compared with wild type. Detailed physiological analyses revealed that NDH deficiency markedly decreased PSI electron transport and CO2 assimilation, particularly under low temperature and sub-saturating irradiance. At moderate and high temperatures, reductions in carbon fixation were largely confined to low-light conditions, whereas at low temperatures, impairment extended across nearly the entire light response range. Under repetitive fluctuating light regimes, NDH-deficient plants showed progressive declines in photosynthesis accompanied by a selective decrease in PSI photochemical capacity without changes in PSII maximum efficiency, indicating PSI-specific photoinhibition. These findings demonstrate that NDH-dependent CET plays a crucial role in sustaining photosynthetic efficiency and crop productivity in dynamic field environments by stabilizing PSI redox balance and maintaining long-term carbon gain. Summary StatementNDH-dependent cyclic electron transport supports photosynthesis and yield in field-grown rice by maintaining PSI function under fluctuating light, low temperature, and sub-saturating irradiance.

Lettuce (Lactuca sativa) is an important field crop, but our understanding of its phenotypic variation and underlying genetics under natural field conditions remains limited, posing challenges for identifying effective crop breeding targets. Longitudinal hyperspectral phenotyping allows for non-invasive monitoring of crop performance under diverse agricultural conditions. In this study, we used hyperspectral imaging to assess the phenotypic variation of almost 200 different field-grown lettuce varieties, following the same plants from just after seedling- to flowering-stage. With automated image processing, we extracted a wide range of spectral phenotypes related to metabolite content, growth efficiency, and environmental stress responses, creating a multi-dimensional time-resolved data set. Principal component analysis (PCA) revealed the major axes of spectral variation over time, and highlighted differences in spectral patterns among lettuce genotypes. Integrating on-site weather data, we modelled GxE interactions of reflectance, revealing regions of the lettuce vegetation spectrum that are primarily shaped by genotype and/or environment. We estimated phenotypic plasticity in response to time, temperature and rainfall using best linear unbiased predictions (BLUPs), capturing genotype-specific developmental trajectories and responses to the environment. We used genome-wide association studies (GWAS) to identify quantitative trait loci (QTLs) of PC-based, single and BLUP-based phenotypes, disentangling the genetic architecture of spectral lettuce phenotypes from major axes of variation down to single wavelength spectral plasticity. These findings provide new insights into the genome-wide genetic regulation and dynamics of spectral phenotypes in field grown lettuce.

Stomatal traits balance carbon gain with water loss, yet their breeding potential in wheat remains underexploited. This study investigated physiological and anatomical stomatal responses alongside yield across two years of large-scale field trials under water-limitation and delayed sowing-induced heat exposure. Across both seasons, stomatal conductance (gs) declined under stress, reflecting strong environmental constraint on gas-exchange (water-limitation: -26.9%; heat: -13.8%). Partitioning responses by leaf surface and genotype identified the adaxial surface as the dominant contributor to gs variation and the most stress responsive. Despite increases in theoretical anatomical gas-exchange capacity (gsmax), gs-efficiency declined, indicating partial decoupling between structural potential and realised conductance. Drought reduced stomatal size while increasing density whereas heat increased size, suggesting stress-specific anatomical plasticity. Moderate-to-high heritability was observed for anatomical traits (Water-limitation: 0.13-0.57; Heat: 0.42-0.71), contrasting with lower and less stable heritability for gs (water-limitation: 0.13-0.41; heat: 0.13-0.50). Genome-wide-association-mapping identified 169 putative QTLs, predominantly for anatomical traits, including stable and co-localised pleiotropic loci. Fourteen sets of closely positioned markers were detected across seasons or studies, with stable regions on chromosomes 2B, 3B and 7B emerging as key loci. Focusing on stable loci controlling adaxial stomatal anatomy offers a realistic strategy to enhance wheat photosynthetic efficiency and climate resilience. HighlightAdaxial stomatal traits dominate gas exchange responses to heat and drought in wheat, with stable anatomical QTL identified on chromosomes 2B, 3B and 7B. Their stability across environments supports their relevance for crop improvement in water-limited and high temperature systems.

Light wavelengths modulate plant growth, metabolism, and physiology. Amaranthus, a C4 underutilized climate resilient crop with promising nutritional properties remained unexplored in terms of metabolite enrichment under monochromatic light wavelengths of visible spectrum. In current study, two cultivars of Amaranthus tricolor (green and red) were exposed to seven light regimes of photosynthetically active radiation (PAR; 400-700 nm): deep blue, blue, green, amber, red, deep red, far red, and their metabolic responses were captured using Gas Chromatography-Mass Spectrometry. The metabolic analysis revealed wavelength-specific reprogramming in the levels of organic acids, sugars, amino acids, fatty acids as well as phenolics. In both the green and red Amaranthus, branched-chain amino acids and phenylalanine, which are nutritionally essential, were significantly elevated under far-red light. While the phenolics such as caffeic acid and ferulic acid were elevated under green and deep blue light respectively in green Amaranthus, amber light wavelengths enhanced these phenolics in red Amaranthus. The study highlighted cultivar-specific metabolic rewiring triggered by specific wavelengths. Altogether, these findings provides insights into metabolic adaptation and demonstrate the ability of light wavelength to specifically enrich the targeted metabolite of nutritional relevance in Amaranthus. It offers strategies to improve the nutritional value of crops in controlled agriculture systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/714947v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1a4477dorg.highwire.dtl.DTLVardef@518550org.highwire.dtl.DTLVardef@7682dorg.highwire.dtl.DTLVardef@4876e2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.