Plant Cell Reports

Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial {sigma} factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5 and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools.

Improving regeneration of ribulose-1,5-bisphosphate (RUBP) is a promising approach to improve photosynthesis and plant growth. In addition to transgenic overexpression of target genes, it could be possible to directly overexpress endogenous target genes, through transcriptional enhancements. As shown by the recent discovery of a short sequence motif, that resembles the known octopine synthase (ocs) enhancer, transcriptional enhancement is achievable by relatively short endogenous sequences. In this study, we query the genome of several model and crop plant genomes for the presence of short enhancer motifs. We find hits across all genomes including some in promoter regions of genes. By using derivatives of these motifs in a transient fluorescence assay, we show that several of these are capable of inducing target gene expression in different promoter contexts. A motif scan of the created constructs, for the presence of known transcription factor binding sites, shows that the insertion of these motifs has created binding sites for different TGA-, NAC- and bZIP-transcription factors. Taken together our study shows the feasibility of finding enhancer sequences in the genomes of different plants. With advancement in gene-editing technologies, like prime editing, using such endogenous enhancer sequences, could allow for precise cisgenic promoter engineering of target genes.

Biolistic transformation is a versatile tool in plant science, yet high equipment costs and tissue damage from high-pressure gas remain significant barriers. Building on our previously developed "TSGMAC", a low-cost, helium-free biolistic system, we report three major advancements to enhance its throughput, delivery quality, and quantitative capability. First, a "guide barrel" assembled from commercial DIY fittings was developed; it effectively eliminates physical tissue damage and ensures uniform particle distribution, even in soft tissues like bok choy (Brassica rapa subsp. chinensis). Second, a rapid gene expression platform using PCR products was characterized. Results demonstrate that linear DNA constructs are efficiently circularized via non-homologous end joining (NHEJ) in plant cells, and protein expression is robust regardless of the relative positions of the promoter, coding sequence, and terminator. This system bypasses time-consuming cloning. Third, a cost-effective, highly sensitive dual-luciferase assay system utilizing teal Luc (teLuc) and inexpensive firefly luciferase (FLuc) inhibitors was established. This integrated workflow enables rapid, quantitative molecular biology using supermarket-obtained materials and standard PCR reagents. Our findings provide a practical foundation for plant scientists, synergistically accelerating gene functional analysis and genetic tool development.

Background and AimsPaspalum notatum reproduces through either sexuality or apomixis, two pathways that may coexist within the same individual and are regulated by interconnected molecular networks responsive to environmental cues. Here, we characterized the transcript structure and expression of BUD SITE SELECTION PROTEIN 13 (BUD13), a component of the RES spliceosomal complex previously reported as differentially expressed in florets of sexual and apomictic plants, as a first step toward testing its involvement in the molecular regulation of the apomixis-sexuality switch. MethodsPreviously generated floral and leaf transcriptomes from sexual and apomictic Paspalum notatum plants, including Oxford Nanopore long-read data, were mined to characterize BUD13 transcript structure and expression. Phylogenetic analyses and in silico mapping were conducted to infer evolutionary relationships and determine the origin of the transcripts. Differential expression was validated by RT-qPCR, while in situ hybridization was used to reveal cell-specific ovule expression patterns. Key resultsBUD13 is expressed in Paspalum notatum florets as a truncated isoform (SHORT) encoding a small protein lacking part of the herpes simplex virus regulatory protein (ICP4) domain. Two SHORT transcripts, SHORT1 and SHORT2, with different 5' untranslated region (UTR) regions, were identified in flowers. SHORT1 was consistently upregulated in apomictic ovules from premeiosis to anthesis. Both transcripts originated from a single genomic locus located in the subtelomeric region of the short arm of chromosome 6. SHORT isoforms with variable structures were detected in other monocots. In situ hybridization showed that, whereas BUD13 was expressed throughout sexual ovules, expression was absent from the female germline of apomictic ovules. A consistent expression was observed in somatic proembryos of aposporous embryo sacs. ConclusionsOur findings reveal structural, spatial and temporal divergence in BUD13 expression between sexual and apomictic reproductive programs, providing new insights into the molecular regulation of asexual seed formation.

Plant regeneration is a major determinant of transformation and genome-editing efficiency, yet the endogenous regulatory networks controlling regenerative competence in horticultural crops remain incompletely understood. The miR319-TCP module regulates multiple developmental processes in plants, but its function in lettuce regeneration has not been defined. Here, we performed a genome-wide analysis of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) gene family in lettuce (Lactuca sativa). Thirty-three LsTCP genes were identified and classified into Class I/PCF, Class II/CIN, and Class II/CYC/TB1 groups. Five CIN-class genes, LsTCP2, LsTCP3, LsTCP4, LsTCP10, and LsTCP24, were predicted as high-confidence miR319 targets and supported by degradome-based cleavage evidence. MIR319-overexpression (OX319) explants showed enhanced de novo shoot regeneration, with 94.5% regeneration efficiency and 1.92 shoots per explant, whereas STTM-miR319 suppression (S319) explants showed reduced regeneration, with 28.5% regeneration efficiency and 0.36 shoots per explant. These phenotypes were associated with altered expression of several miR319-targeted CIN-TCP genes, particularly LsTCP4, LsTCP10, and LsTCP24. Disruption of LsTCP4 increased regeneration efficiency to 91.4% and shoot production to 2.05 shoots per explant, resembling the regeneration-enhancing effect of miR319 overexpression. In contrast, disruption of the non-target CIN gene LsTCP17 did not significantly affect regeneration under the tested conditions. Together, these results identify LsTCP4 as a key miR319-responsive negative regulator of de novo shoot regeneration and highlight miR319-mediated repression of LsTCP4 as a potential endogenous strategy for improving lettuce regeneration.

Hemp (Cannabis sativa) produces a wide array of medicinally significant compounds, including cannabidiol (CBD). These compounds are predominantly synthesized in female hemp inflorescences. The proposed research utilizes next-generation sequencing-based transcriptome analysis using a 3{square}-end-directed approach to identify differentially expressed genes between male and female hemp plants at the early vegetative stage. 886 differentially expressed genes (DEGs) were identified, a majority of which were upregulated in males compared to females. We hypothesized that alternative RNA processing contributes to sex-specific gene expression. To this end, 932 genes were identified that exhibited significant changes in poly(A) site usage when comparing males and females. These genes were much more likely to be differentially expressed, supportive of this hypothesis. Males tend to have longer 3 UTRs with canonical motifs found in the Near-Upstream Elements (NUE), compared to the shorter 3 UTRs in females, which have A-rich motifs near the cleavage site. This suggests that polyadenylation remodels hemp mRNAs with distal poly(A) sites being preferred in males. To further investigate when this sex-specific gene expression program is established, RNA was isolated from plants at various developmental stages, such as developing seeds, four-day-old seedlings, and different developmental stages up to four weeks after sowing. Diagnostic male-specific genes were analyzed using RT/PCR. The results indicate that sex-specific gene expression is not evident in seeds but rather is set during or after germination. SignificanceO_LIHemp males tend to have longer 3 UTRs with canonical motifs found in the Near-Upstream Elements (NUE), compared to the shorter 3 UTRs in females, which have A-rich motifs near the cleavage site. C_LIO_LIThe sex-specific gene expression program is not yet established in mature seed but is set in the time between germination and 4 days of growth. C_LI

O_LIEngineering nitrogen fixing crops requires not only transferring the nitrogenase structural genes, but also the accessory genes to synthesize its iron-sulphur cofactors. Scaffold protein NifU is a critical element in this system as the starting point of nitrogenase cofactor assembly. NifU has been successfully produced in plants, however, its optimal production required high levels of iron in the medium. This is likely due to a faulty connection with the endogenous iron trafficking network C_LIO_LITo identify specific elements targeting iron to NifU, pull-down assays were performed to identify showing bacterioferritin A (BfrA) as a likely candidate. Co-immunopurification, mutant characterization, iron transfer assays, and co-expression in Nicotiana benthamiana assays were carried out. C_LIO_LIBfrA transfers iron to NifU through protein-protein interactions. When these two proteins were co-expressed in N. benthamiana leaves, there was an increase in NifU production. In turn, it led to doubling NifH synthesis, a nitrogenase structural protein that is also required for the synthesis of the more complex nitrogenase cofactors. C_LIO_LIOur results provide a new element towards engineering nitrogen-fixing crops. They also underscore the importance of transferring the metal delivery systems when expressing metalloproteins in heterologous systems. C_LI

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1-13.8% for NbPDSa and 7.6-23.0% for NbPDSb, while lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, indicating that heritable editing was not achieved under these conditions. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

Plant hormones regulate almost every aspect of plant growth and development. KARRIKIN INSENSITIVE 2 (KAI2)-dependent signaling, which is thought to transduce signals derived from an unidentified ligand known as the KAI2 ligand (KL), regulates numerous traits, including seed germination in angiosperms and vegetative reproduction in bryophytes. The origin of KAI2 is believed to be ancient, and the evolution of its signaling pathways remains of significant interest. Ferns represent critical lineages for elucidating the evolution of land plant traits and growth mechanisms that enabled adaptation to terrestrial environments. Therefore, functional studies of key components of this pathway in ferns are essential for understanding the evolutionary trajectory of KAI2-dependent signaling during vascular plant diversification. However, experimental platforms for the CRISPR/Cas9 system, a powerful tool for investigating gene function, remain undeveloped in ferns. Here, we report an efficient CRISPR/Cas9 system based on ribozyme-gRNA-ribozyme (RGR) technology in the model fern, Ceratopteris richardii (C. richardii). We generated loss-of-function mutants of the KAI2 ortholog in C. richardii (CrKAI2), as well as the signaling components CrMAX2 and CrSMXL. We demonstrate that exogenous application of an artificial KL agonist increases the expression of KAI2-dependent signaling responsive genes in wild type plants; this response is abolished in Crkai2 mutants. These findings indicate that KAI2-dependent signaling is conserved in C. richardii. Furthermore, this study proposes an efficient CRISPR/Cas9 method that will facilitate genetic studies in ferns.

Long non-coding RNAs (lncRNAs) have emerged as critical players in plant development and stress responses, yet their involvement in pollination responses is largely unknown. To address this gap, we identified and characterized lncRNAs and their cis-acting, trans-acting, and miRNA-mediated regulatory interactions during both compatible and incompatible pollination in Arabidopsis thaliana. Leveraging publicly available datasets, we analyzed expression profiles at 10 and 60 minutes post-pollination. We identified 1,073 novel and 3,422 annotated lncRNAs, with 1,002 novel and 985 annotated, respectively, showing detectable expression after filtering. Differential expression analysis identified 12 lncRNAs at 10 min and 32 lncRNAs at 60 min post-pollination. Further investigation revealed 9 cis-targets, 112 trans-targets, and 144 miRNA-mediated regulatory interactions, many of which were enriched in pathways related to stress, defense, and self-incompatibility. Notably, the regulatory landscape is more active at 60 minutes than at 10 minutes post-pollination. These findings provide a robust framework and resource to facilitate future functional studies of lncRNAs during pollination.

Low temperature stress causes significant damage to the strawberry plant. During cold stress, plants undergo morphological and physiological changes often regulated at the genetic and/or epigenetic levels. Some strawberry cultivars are more cold-hardy than others. Using the diploid woodland strawberry as a model, we analyzed the effects of cold acclimation on methylome and transcriptome dynamics in the crowns and leaves of three ecotypes with contrasting cold tolerance. Alta, which was the most cold-tolerant ecotype, exhibited the highest genetic and epigenetic plasticity in response to cold. CHH-context methylation dominated the differentially methylated regions (DMRs) with more hypomethylation in crowns and hypermethylation in leaves. CG methylation was enriched in gene bodies, while non-CG methylation was prevalent in upstream and downstream regions. Our study revealed that less than a quarter of differentially methylated genes (DMGs) showed changes in transcript accumulation levels. This finding indicates that universal cold response in Fragaria vesca, as reflected by gene expression, cannot be mechanistically attributed to DNA methylation. The majority of differentially expressed differentially methylated genes (DEDMGs) were ecotype- and tissue-specific. Enrichment analysis revealed that these genes were involved in pathways related to stress tolerance, such as carbohydrate metabolism, lipid metabolism, ATP hydrolysis, and cellular detoxification. Each ecotype responded to cold through mobilization of its own set of differentially expressed genes (DEGs), DMGs, and DEDMGs, and variation in expression and methylation patterns exhibited by Alta, FDP817, and NCGR1363 suggest that cold signaling processes and survival depend on the tissue, ecotype, and geographical origin of the plants exposed to cold stress. Therefore, this study highlights the potential of both genetic markers and epialleles as molecular markers for the development of cold-tolerant octoploid strawberry cultivars that are better suited for propagation in Nordic climates.

BackgroundUrtica dioica, also known as stinging nettle, is a widespread plant that can indicate high nitrogen availability in the soil. It is probably best known for the pain caused by touching it. U. dioica is also recognized as a medicinal plant with reports claiming applicability against numerous diseases. ResultsA highly continuous genome sequence was constructed based on nanopore long read sequencing data. The total assembly size is 1.1 Gbp with an N50 of 40.7 Mbp. RNA-seq data and hints from other species were integrated to produce a high quality annotation of the protein encoding genes. This genomic resource enabled the identification of genes involved in the flavonoid biosynthesis. A particular focus was on anthocyanin biosynthesis genes as these are crucial for high light and nitrogen deprivation stress response, which is revealed by redding of the leaves. ConclusionThis genomic resource provides the basis for future studies unraveling the biosynthesis pathways underlying various medically important compounds produced by stinging nettles.

The biotrophic pathogen Puccinia triticina is the causative agent of the most vulnerable foliar disease, namely leaf rust disease of wheat. The pathogen-secreted effectors are essential in modulating fungal virulence and host immune responses. Despite their significance, potential effectors and their underlying mechanisms governing host susceptibility remain elusive. In the present study, we employed an in silico approach to identify and characterise effector proteins from the P. triticina proteome. Later, performed temporal expression profiling to prioritise effector candidates associated with rust disease. Here, a total of 273 high-confidence effector candidates were identified and analysed their physicochemical properties, domains, motifs, and functional annotations, to assess their conservation and dynamics. Although most of the effectors were uncharacterised, the conserved motif virulence-associated [YFW]xC was notably enriched in the effector repertoire. Comparative PHI-base annotation highlighted similarities with known fungal virulence factors involved in host susceptibility. Effectors harbouring CAZyme activity indicate involvement in host cell wall modification. Promoter analysis identified multiple stress- and defence-related transcription factor binding sites, suggesting regulated expression during infection. Transcriptome analysis revealed that 20 effector genes were significantly upregulated during P. triticina infection. qRT-PCR validated the expression of 4 highly induced effector transcripts following P. triticina infection in susceptible wheat variety. Specifically, two of these candidates demonstrated biphasic expression pattern that aligns contrasting PTI- and ETI-mediated defense mechanisms critical for sustained virulence. Overall, this study provides a comprehensive framework for identifying functionally relevant P. triticina effectors and offers insight for future effector-target studies and effector-based leaf rust management strategies.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

Elongating rice internodes present a developmental gradient from dividing meristem to mature cells, providing an elegant pseudo-time course for study of plant vegetative development. We tested the hypothesis that DNA methylation regulates gene expression during rice internode development by integrating RNA-seq and bisulfite DNA sequencing across eight internode segments. Previously described topologically associated chromatin domain borders aligned with transcription start sites of constitutive expressed genes. CpG and CHG differential methylation was enriched in young segments, consistent with maintenance methylation; whereas CHH methylation showed similar differential abundance in young and old segments. CHH and CHG methylation in upstream regions, CpG methylation within gene bodies, and any methylation in 5' and 3' untranslated regions were permissive of moderate to high gene expression. Very low expression was associated with CpG methylation upstream, CHG and CHH methylation within gene bodies, and CpG and CHG methylation downstream. A nonrandom subset of genes, including cell wall-related glycoside hydrolases, lignin and tricin biosynthesis enzymes, and WD40 proteins, showed methylation-expression correlations, with expression changes enriched in triple-marked elements. These results suggest that internode phenotypes of DNA methylation machinery mutants relate to alteration of specific target genes, opening approaches for grass culm improvement for lodging resistance and biomass production.

In vitro regeneration of Citrus plants is a widely used method, however, induction of adventitious roots from regenerated shoots remains a major bottleneck, limiting the recovery of healthy plants for commercial production and genomic research for crop improvement. We established an in vitro regeneration system producing profuse, healthy roots for sweet orange (Citrus sinensis cv. Benyenda) by optimising combinations and concentrations of auxins. Prior to optimising the rooting media (RTMs), we obtained a shoot regeneration rate of 90.6% from sweet orange epicotyl explants using a cytokinin, 6-benzylaminopurine (BAP). Across twelve auxin-supplemented RTMs containing different concentrations of indole-3-butyric acid (IBA) and/or 1-naphthaleneacetic acid (NAA), rooting percentages ranged from 8 - 87.5%. The combination of IBA 1.0 mg L-1 and NAA 0.1 mg L-1 promoted the best overall performance, 75 {+/-} 7.2% rooting percentage with healthy, callus-free roots ([≥]5 cm in length), whereas other RTMs with other auxin combinations induced callus and limited root elongation. The best-performing SRM and RTM were subsequently used for selection and recovery of transgenic sweet orange lines carrying an empty CRISPR/Cas9 construct, resulting in an 4.8% transformation efficiency. Both transgenic and non-transgenic rooted plantlets were successfully acclimatised under glasshouse conditions with a survival rate of 90%. This enhanced regeneration system overcomes rooting bottleneck and improves plant survival,enabling faster recovery of transgenic citrus lines within four months. It supports accelerated development for commercial applications and advances in citrus genetic improvement.

Ovate Family Proteins (OFPs) are a class of plant-specific, negative nuclear transcriptional regulators characterized by conserved C-terminal OVATE domain. This study on comparative functional characterization of two head-to-head arranged OFPs - AtOFP2 (Ovate-OFP with full ovate domain) and AtOFP17 (Ovate-Like OFP with partial ovate domain) provides critical insight into how structural variations in ovate domain leads to functional divergence. Detailed phenotypic analysis of 28 physical and physiological traits of loss- and gain-of-function mutants revealed that both genes act as broad, pleotropic repressors of plant growth and development. Removal of repression in knock-down mutants of both genes exhibited reduced duration of seed dormancy, faster rate of germination and growth, bigger plants and significantly higher seed yield. In contrast, constitutive over-expression showed a generalized repressive nature of both genes, with nuanced differences for fine tuning of specific traits. For example, both genes showed antagonistic behaviours on root hair architecture. AtOFP2 act as a strong repressor of root hair development whereas AtOFP17 is a stronger repressor of hypocotyl and root cell architecture. AtOFP17 owing to partial ovate domain exerts a mild level of repression throughout life span as indicated by smaller plants and lesser yield in knock-down AtOFP17 mutants. On the contrary, AtOFP2 exerted a much stronger repressor effect in which > 90% over-expression mutants died at the juvenile stage ; the survival of remaining 10% is probably owing to activation of dosage-dependent feedback loop mechanism as indicated by normal growth of mature plants, and is also evident by transcriptome data. Transcriptome analysis of roots of 7-day old seedling of knock-down and over-expression mutants of AtOFP2 showed downregulation of OFP2 in over-expressed mutants. However, severely stunted phenotype indicated presence of stable OFP2 protein to exert effects. Analysis of DEGs in OFP2 mutants revealed that it acts as an important regulator working at intersection of hormonal signalling affecting critical genes required for auxin, cytokinin, GA, BR and ABA functioning. Perturbations across hormonal signalling pathways affects cell wall remodelling factors such as EXPANSINS, Xyloglucan hydrolases (XTHs) and cellulose synthases (CSLs) causing overall stunted growth; and epidermal patterning genes such as WER, GL1, EGL3, TTG1 leading to severely reduced root length and root hairs. Significantly, functional analysis of this master regulator highlighted a significant economic potential. Knockdown of both these genes relieves their natural repression on reproductive traits, leading to longer siliques, bigger and heavier seeds, and substantially increased overall seed yield, positioning AtOFP2 and AtOFP17 as highly valuable targets for agricultural crop improvement.

The production of reactive oxygen species (ROS) in response to cadmium (Cd) has been extensively studied, demonstrating that they play a key role in the plants response to this heavy metal. While the role of enzymes like RBOHs has been thoroughly studied, the function of other ROS-producing enzymes, such as peroxisomal glycolate oxidase (GOX), remains largely overlooked. Peroxisomal GOX is a core metabolic enzyme of the photorespiratory pathway occurring in chloroplasts, mitochondria and peroxisomes. Using Arabidopsis (Arabidopsis thaliana) mutants lacking the main peroxisomal GOX genes, GOX1 (gox1-1) and GOX2 (gox2-1) we explored their function in plant response to Cd. Although photosynthetic capacity appears to be affected to the same extent in both mutants under control and Cd stress conditions, GOX2 seems to play a greater role in ROS production in response to the metal. Transcriptomic analyses on WT and gox2-1 pointed to the mitochondrial electron transport chain (mETC) as a target of Cd stress. We further investigated the individual GOX1 and GOX2 functions in mETC regulation and redox state. Although oxidative ratio of mitochondria was higher in both mutants, it was more pronounced in the absence of GOX1. Furthermore, the mETC is affected in both mutants but the regulation of its components differs in each mutant. These results point out the different functions of the two photorespiratory GOX isoforms in Arabidopsis, leading to a better understanding of the photorespiratory pathway.

BackgroundTransposable elements (TEs) are mobile genomic sequences that constitute one-third to one-half of the mammalian genome. Recently, TEs have been recognized for their important roles as cis-regulatory elements. TEs are broadly activated during zygotic genome activation (ZGA) in mammalian embryos, where they function as alternative promoters of host genes and drive the transcription of chimeric transcripts. However, the construction of comprehensive chimeric transcript databases based on short-read sequencing remains limited due to the repetitive and abundant nature of TEs in the genome. Here, we used long-read RNA sequencing to construct a comprehensive dataset of chimeric transcripts expressed in ZGA mouse and bovine embryos. ResultsWe identified 11,996 and 4,755 chimeric transcripts variants derived from 2,695 and 1,200 host genes in mouse and bovine, respectively, exceeding the numbers reported in previous short-read-based studies. Among them, 114 orthologous pairs produced chimeric transcripts in both species. Gene Ontology analysis revealed significant enrichment of terms related to transcriptional regulation and protein modification in mouse, whereas no terms were significantly enriched in bovine. Assessment of the protein-coding potential of the TE-driven transcripts using predicted open reading frames (ORFs) revealed that the proportion of "Protein-coding" transcripts was lower, whereas that of "LncRNA" (long non-coding RNA) was higher compared with all transcripts in both species. Among the ORFs classified as "Protein-coding", comparison with canonical ORFs revealed a tendency for the N terminus to be truncated while the C terminus remained intact in both species. TE-derived promoters used in mouse were enriched for mouse-specific TEs, whereas those in bovine were enriched for older TEs conserved among eutherians. In addition, long-read sequencing detected a greater number and proportion of TEs used as promoters in mouse and bovine than short-read sequencing. Although motif analysis identified KLF5 and OTX2 binding sites upstream of TE-derived promoters in both species, the specific TEs containing these motifs differed between the two species. ConclusionsThis study presents the first long-read sequencing analysis of chimeric transcripts in mammalian embryos in two species. Our approach revealed the functional similarities of chimeric transcripts between species, as well as species-specific differences in their TE compositions.

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.