Plant Cell Reports

Phtheirospermum japonicum is a genetic model for parasitic Orobanchaceae, a plant family that includes noxious parasitic weeds from the genera Striga, Orobanche, and Phelipanche (Ishida et al., 2011). Striga species alone cause billions of dollars in annual losses by reducing yields of major crops (Pennisi, 2010). The lack of stable transgenesis protocols often hinders heritable CRISPR/Cas9 genome editing for gene function analysis in crops and species beyond standard model plants, including parasitic Orobanchaceae (Steinberger and Voytas, 2025). Here, we adapted a virus-mediated delivery system for ultracompact TnpB nucleases, enabling genome editing independently of tissue regeneration or floral dip transformation in the parasitic plant P. japonicum (Nagalakshmi et al., 2025; Weiss et al., 2025).

BackgroundMaize knobs are regions of constitutive heterochromatin that are readily identified in both meiotic and somatic chromosomes. These structures have been characterized as stable throughout the cell cycle, exhibiting late replication during the S-phase, and are composed of two specific families of highly repetitive DNA sequences: K180 and TR-1. Although widely used as cytogenetic markers due to their variability in number and chromosomal position across inbred lines, hybrids, and landraces, little is known about their chromatin structure and dynamics. In this study, we analyzed chromatin accessibility of knobs using DNS-seq data across four maize tissues representing distinct developmental stages. ResultsOur results reveal that K180 knobs exhibit tissue-specific variation in chromatin accessibility, transitioning between open and closed states during development. In contrast, the TR-1 knob of chromosome 4 remained consistently inaccessible across all tissues analyzed. A knob composed of both K180, and TR-1 further supported this observation, with only the K180 region showing dynamic accessibility. To validate these findings, we also analyzed other repetitive regions such as centromeres, which showed a uniformly closed chromatin structure similar to TR-1. These results suggest a unique developmental modulation of chromatin accessibility associated with K180 repeats. While the chromatin accessibility of knobs does not reach the levels observed at Transcription Start Sites (TSS), the comparison among different classes of repetitive DNA within maize constitutive heterochromatin provides compelling evidence for sequence-specific and tissue-specific chromatin dynamics. ConclusionsOur findings uncover a previously unrecognized property of maize knobs and establish a reference for future studies on chromatin organization and epigenetic regulation of repetitive DNA in plant genomes.

Triple-negative breast cancer (TNBC) remains one of the most aggressive breast cancer subtypes and lacks durable targeted therapies. Dysregulation of cell-cycle control, particularly through CDK4/6 signaling, is a defining feature of TNBC biology (Garrido-Castro et al., 2019). Extracts of Moringa oleifera have repeatedly been shown to induce G1-phase arrest in breast cancer models, yet the molecular basis of this phenotype remains unclear (Al-Asmari et al., 2015) (Gaffar et al., 2019). Emerging work on cross-kingdom regulation has raised the possibility that plant-derived microRNAs may, under specific conditions, interact with mammalian transcripts (Zhang et al., 2012) (Chin et al., 2016). Sequence shuffling for the negative control was performed with set.seed(42) to ensure reproducibility. Additional visualisations (nucleotide alignment and thermodynamic analyses) were generated using Python 3 (matplotlib v3.7). Here, we performed a high-stringency computational screen of conserved Moringa microRNAs against 30 genes implicated in TNBC pathogenesis using local sequence alignment. We identify a predicted high-affinity interaction between mol-miR156 and the human CDK4 3' untranslated region (3'UTR), characterized by an uninterrupted 12-nucleotide complementary motif that exceeds canonical mammalian microRNA seed requirements. These findings support the hypothesis that conserved plant microRNAs may exhibit latent structural compatibility with oncogenic human transcripts. While physiological delivery and functional repression are not demonstrated here, this work establishes a molecular framework for future experimental investigation into cross-kingdom RNA interactions relevant to cancer cell-cycle regulation. Impact StatementA high-stringency computational screen identifies latent molecular compatibility between a conserved plant microRNA and the human CDK4 oncogene, establishing a testable framework for cross-kingdom RNA interference in triple-negative breast cancer.

Understanding the regulation of enzyme activity involved in photosynthesis is essential for engineering enhanced carbon fixation in crops. In C4 plants, the enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is one of the most abundant leaf enzymes and plays an essential role in photosynthetic carbon dioxide (CO2) fixation. The enzyme also plays a key role in central metabolism (e.g., providing intermediates to the citric acid cycle) and therefore must be highly regulated to coordinate its activity. The regulation of PEPC activity can occur allosterically by glucose 6-phosphate activation and malate inhibition, which is in part influenced by reversible phosphorylation. A specific light-dependent phosphorylation of PEPC at an N-terminal serine residue by the PEPC-protein kinase (PEPC-PK) can regulate its sensitivity to this allosteric regulation. However, the impact of this PEPC phosphorylation has not been tested in a C4 crop. Therefore, we created PEPC-PK mutant lines in Zea mays to assess the impact of PEPC phosphorylation on its allosteric regulation, photosynthesis, and growth. While the maximum PEPC activity was unchanged, PEPC in the PEPC-PK mutant plants was not phosphorylated under light and was more sensitive to malate inhibition. However, gas exchange, electron transport, and field biomass analyses showed no differences in the PEPC-PK mutant plants. These results demonstrate that in Z. mays PEPC phosphorylation affects enzyme sensitivity to malate in vitro but does not substantially alert photosynthetic performance or growth under field conditions suggesting additional regulation of PEPC activity in planta.

Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprises the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompass most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (genus Potyvirus)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings must be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.

Plant genomes are filled with retrotransposons and their derivatives, subject to constant sequence turnover. As short, non-autonomous retrotransposons do not encode a protein product, they experience reduced selective constraints on their DNA sequence, leading to diversification into multiple families, usually limited to only a few species. This absence of any coding capacity and their tendency to form subfamilies are the reasons for the incomplete description of non-autonomous LTR retrotransposons in most to all genomic repeat annotations. Here, we focus on non-autonomous LTR retrotransposon identification. Are all of these sequences derivatives of easier-to-identify full-length elements? Or is there more variability, which is currently overlooked? For this, we capitalize on our comprehensive understanding of the TE landscape in sugar beet to assess the extent of the blind spot on non-autonomous LTR retrotransposons Here, we present a workflow to identify non-autonomous LTR retrotransposons without prior sequence information, retrieving more than 100 families within the sugar beet genome. We only include TEs without the ability for complete self mobilization. Spanning up to 15,000 bp, these non-autonomous families are often longer than expected and characterized by reshuffling and modular evolution. Most strikingly, only a few of these families are directly derived from autonomous partners, showing that there is a large, undiscovered TE variety in the non-autonomous TE fraction. We highlight that a large fraction of non-autonomous TEs wont be retrieved with the current TE identification workflows, even if the output is well-curated and condensed into TE libraries and suggest procedures to remedy this gap. This study is the first insight into the non-autonomous LTR retrotransposon landscape within a single genome and serves as an example to estimate the error in non-autonomous TE detection.

RNA interference (RNAi) shows great potential to protect crops against fungal diseases, yet reported protection efficiencies vary greatly, and our understanding of the factors responsible for this variance remains limited. In this meta-analysis, we evaluated 89 studies that compare the efficiency of host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS) in controlling fungal diseases, focusing on biotrophic, hemibiotrophic, and necrotrophic fungi, the use of formulations, and the dsRNA design as explanatory factors for differences between reported efficiency values. Our results indicate that SIGS is slightly more effective, particularly in biotrophs. Surprisingly, SIGS studies using formulations did not outperform those applying naked dsRNA. We also assessed parameters of RNA design. Differences in dsRNA length and the number of constructs, and number of targets showed no consistent significant effect on resistance in either HIGS or SIGS. Interestingly, however, HIGS studies reported significantly higher efficiency when targeting genes closer to the 3 end and SIGS when targeting genes closer to the 5 end. We discuss potential reasons for the reported patterns, such as variability in dsRNA uptake mechanisms, intercellular trafficking and Dicer processing, and conclude that more research is needed to understand the biological mechanisms determining RNAi efficiency for fungal control.

The model plant species Arabidopsis thaliana has two nucleolus organizer regions (NORs), each consisting of tandem repeats of 45S rRNA genes on chromosomes 2 (NOR2) and 4 (NOR4). In the ecotype Col-0, the rRNA gene subtypes mapping to NOR2 are mostly silenced during development, whereas those mapping to NOR4 are mostly active. Using molecular and genetic mapping approaches, we demonstrate in multiple epigenetic mutants the occurrence of NOR conversions involving loss of rDNA and the associated telomeres in one NOR and replacement of the lost sequences with the corresponding sequences from the other NOR. We studied mutants of evolutionarily conserved chromatin remodelers DECREASE IN DNA METHYLATION 1 (DDM1) and CHROMATIN ASSEMBLY FACTOR 1 (CAF-1), and plant-specific CHROMOMETHYLASE 2 (CMT2) DNA methyltransferase. We observed NOR conversions in ddm1 and caf-1 mutants, where such NOR conversions altered the rRNA variant expression patterns, which often confound the data pertaining to release of rRNA gene silencing. We delineated the effects of these mutations on the rDNA instability-mediated alterations in rRNA variant expression from their effects on the actual release of rRNA gene silencing. We also show that these mutations release rRNA gene silencing independently of their effect on rDNA genomic instability involving NOR conversions.

Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing (Hu et al. 2025; Liu et al. 2025; Weiss et al. 2025). We recently established a VIGE approach for heritable editing at single loci in Arabidopsis by delivering the compact genome editor ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV) (Weiss et al. 2025). Here, we greatly improved this approach by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) (Zhou et al. 2026) to develop an efficient multiplexed genome editing platform.

Virus induced gene silencing (VIGS) is a method that exploits plant antiviral defence mechanisms to downregulate endogenous genes. The technique is versatile, rapid, and widely used for functional genomics studies. Here we report a method for VIGS in the medicinal plant, Calendula officinalis (pot marigold). This species produces anti-inflammatory triterpenoids and has also been bred and cultivated as an ornamental plant. We describe a method for the injection of Agrobacterium tumefaciens cultures into leaf midribs and compare visual marker genes for tracking VIGS utilising constructs that simultaneously target visual marker and target genes. We use these tools to demonstrate that silencing a gene encoding cycloartenol synthase results in changes to leaf phytosterols. This method could be used to further investigate the genetic basis of specialised metabolism in this species and could be adapted to other members of the Asteraceae family, many of which are of economical and chemical value.

Published studies have reported species-variance between profiles of Calvin-Benson cycle (CBC) intermediates, not only between C4 species and C3 species, but also within C3 species (Arrivault et al., 2019, Borghi et al. 2019). It was proposed that this variance reflects lineage-dependent changes in the balance between different reactions, or poising, of the CBC. These earlier studies investigated phylogenetically-unrelated C3 species. In the current study, CBC intermediates were profiled in five closely-related species from Solanum sect. lycopersicon subsect. Lycopersicum. The levels of individual CBC intermediates showed many significant differences. In a principal component analysis, whilst three species (Solanum lycopersicum, Solanum cheesmaniae, Solanum neorickii) overlapped, Solanum pimpinellifolium and especially Solanum pennellii grouped separately, and were at opposing ends of the distribution. When combined with published data, whilst the separation between Solanum species was retained, they formed a group that was separated from five other C3 species, as well as two C4 species. It is discussed that the observed variation in CBC metabolites profiles within Solanum, together with their separation from other C3 species, supports the idea that CBC evolution is shaped both by phylogenetic relatedness and lineage-specific adaptation. HighlightVariance of intermediate levels points to poising of the Calvin-Benson cycle varying between closely-related species in the tomato clade Solanum sect. lycopersicon subsect. Lycopersicum

In this study, the level of Rubisco protein was reduced in wheat using RNAi, to test the hypothesis that photosynthesis, growth, and grain yield could be maintained whilst improving nitrogen use efficiency. The RNAi Rubisco wheat plants, with a Rubisco activity of less than 70% of wild type (WT) plant levels, had reduced photosynthesis, reductions in leaf and stem biomass and decreased seed yield. Interestingly, in the wheat RNAi Rubisco lines that had a small (<30%) reduction in Rubisco activity, the seed number, total seed weight and harvest index were comparable to that of WT type plants. However, no improvement in photosynthetic nitrogen use efficiency (PNUE) was evident in any of the RNAi Rubisco lines. Notably, PNUE was lower than for WT wheat plants in the RNAi lines with more than a 30% reduction in Rubisco activity. This result was unexpected and caused by an accumulation of N in both the leaves and seeds. At present we do not have an explanation for this but one possible hypothesis is that it could be due to slower growth caused by a reduction in source strength in the RNAi plants, which in turn resulted in changes to carbon and nitrogen allocation. HighlightWheat RNAi plants with small reductions in the amount and activity of Rubisco had a similar biomass and total seed weight to that of untransformed controls but no improvement in nitrogen use efficiency was evident.

BackgroundRAB Guanine Nucleotide Dissociation Inhibitors (RAB GDIs) are important vesicle transport regulators in eukaryotes, participating in the functional cycle of RAB GTPases by stabilizing their non-active GDP-conformation. AimsWe address the importance of the three Arabidopsis thaliana RAB GDI paralogs by genetic and developmental analyses and put these results into the seed plants evolution context. MethodsWe use methods of genetics, microscopy and phylogenetics. ResultsOur genetic analyses of Arabidopsis T-DNA insertional mutants confirm recent CRISPR alleles data indicating lethality of double gdi1 gdi2 mutants, and our microscopic data point to embryo development arrest in double mutant seeds. We also confirm the involvement of GDI2 and GDI3 in pollen tube growth. Moreover, our data show that GDI1 also contributes to proper pollen function. Our phylogenetic analysis reveals independent diversification of RAB GDIs in Gymnosperms and Angiosperms, with early specialization of an Angiosperm reproduction-and gametophyte-related clade. ConclusionsIn Arabidopsis, RAB GDI1 and 2 are important for the vegetative growth while RAB GDI2 and 3 are vital for reproduction. Evolution of the RAB GDI family reflects the evolution of seed plants. HighlightsRAB GDIs are vital for plant growth and reproduction and act redundantly. Even the low-transcribed RAB GDI1 isoform contributes to the proper pollen function. Two RAB GDI clades evolved in early Angiosperms.

Improving rice (Oryza sativa L.) yield requires a balanced enhancement of both sink size and source capacity. While many QTLs for sink size have been identified, only a few are known for source capacity, which is essential for achieving high yield. Here we identified qHP10 as a major QTL for increased photosynthetic rate by using chromosome segment substitution lines derived from a cross between the high-yielding indica cultivar Takanari and the average-yielding japonica cultivar Koshihikari. High-resolution mapping combined with CRISPR/Cas9-induced mutagenesis revealed that the causative gene underlying qHP10 is Mitogen-Activated Protein Kinase 4 (OsMPK4). A near-isogenic line carrying the OsMPK4Takanari allele (NIL-OsMPK4) had a 15-25% higher photosynthetic rate than Koshihikari. NIL-OsMPK4 also had higher stomatal conductance than Koshihikari but similar stomatal pore size and density, indicating that increased stomatal aperture increases photosynthetic rate. This enhancement is likely attributable to the down-regulation of OsMPK4 expression, which increases stomatal conductance and thus promotes CO2 uptake. Our findings demonstrate that OsMPK4 is a promising genetic target for increasing source capacity and, potentially, rice yield through molecular breeding. (175 words)

Particle bombardment systems are widely used for plant transformation, but commercial devices are expensive and rely on high-pressure helium gas. This study aimed to develop a cost-effective and helium gas-free alternative using an air duster gun connected to a commercial compressor. A nozzle (for DNA with transgenes), gold particles (as DNA carriers), nozzle-to-sample distance, and a method for coating gold particles with DNA were optimized to yield better transformation efficiency in targeting onion epidermal cells and rice calli. From the rice calli transformed with the newly developed system (a tool to shoot genes with massive air from a compressor: TSGMAC), stable transgenic plants could be obtained. TSGMAC offers a low-cost and helium gas-free solution for plant transformation and genome editing and can enhance accessibility to particle bombardment-based techniques.

Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report an improved protocol for protoplast isolation from seedling leaves and demonstrate, for the first time, transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 {+/-} 6.19%. Transformation efficiency was strongly influenced by season, which also affected protoplast yield. Both, a basic molecular toolkit for functional genomics and future biotechnological applications were established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). The establishment of a reliable protoplast transformation and editing system provides a crucial foundation for future genetic improvement and functional studies in this non-model tree species.

Peanut (Arachis hypogaea L.), a vital oilseed and food legume, is cultivated across the globe. Genetic improvement via conventional breeding faces limitations from narrow diversity and reproductive barriers, underscoring the need for tissue culture-based regeneration and transformation platforms. This study optimizes an efficient, reproducible in vitro regeneration protocol using cotyledonary node explants from three Indian elite cultivars: GG-20, GJG-9, and TAG-37A. Explants from aseptically germinated seedlings were cultured on Murashige and Skoog (MS) medium with varying cytokinins (e.g., BAP 0-4 mg/L) and auxins (e.g., NAA 0.1-0.9mg/L), yielding direct multiple shoot induction without callus, minimizing somaclonal variation. Optimal shoot proliferation occurred on full-strength MS + 2 mg/L BAP for GG-20/GJG-9 (88.9% efficiency) and 4 mg/L BAP for TAG-37A ([~]89-100% efficiency); rooting peaked on half-MS + NAA (up to 88.9% in GG-20). Regenerated plants acclimatized successfully in greenhouse conditions. Additionally, a robust in planta Agrobacterium tumefaciens (EHA105, pGFPGUSPlus) transformation via plumular meristem pricking in GG-20 achieved 7.69% efficiency. Transgene integration was confirmed by GUS assay and PCR (GUS/hptII), with [~]64% soil establishment.

The nucleus is the characteristic organelle for eukaryotic organisms. Unlike the classic textbook view of static two-dimensional nuclei, nuclear shape is dynamic inside the live cell. The alteration or deformed nuclear shape is the hallmark of cancer in animal cells and environmental stress in plants. The nuclear envelope proteins interact with chromatin to regulate gene expression. Unfortunately, we have limited knowledge about the impact of abiotic stress on nuclear shape, movement, and chromatin dynamics. To circumvent this issue, we are utilizing a dual fluorescently tagged marker lines - nuclear envelope protein and chromatin - to perform live cell imaging in the model plant Arabidopsis thaliana root. The live cell imaging was performed in control and salt-stressed conditions. We utilized these captured movies to analyze through open-source image processing software Fiji/ImageJ with the help of the TrackMate plugin. Using this method, we have demonstrated that chromatin velocity is decreased in salt-treated conditions. This method will be widely applied to quantitative live cell imaging of nuclear shape and chromatin dynamics during plant development and environmental stress. SummaryThis process aims to simultaneously record nucleus and chromatin dynamics in Arabidopsis thaliana roots and investigate changes in these dynamics in response to developmental and environmental cues.

Short Interrupted Repeats Cassettes (SIRC) are recently discovered eukaryotic DNA elements possessing many traits of satellite DNA and mobile genetic elements, and consisted of short direct repeats interspersed with diverse spacer sequences. The SIRC ensemble of individual species is highly heterogenous and cannot be studied using alignment methods. It was found that number of similar SIRC sequences in a given pair of species is in general correlated with their taxonomic distance, and, at the same time, closely related species can possess very diverged SIRC ensembles, which makes SIRC evolutionary pattern closer to mobile genetic element type. The SIRC sequences make up clusters with comparable sequence patterns, that are likely to demonstrate doublet evolutionary model which strongly supports that the SIRC structure is supported by the evolutionary selection. Several SIRC sequences of Arabidopsis were found to be of ancient origin with traceable evolution history as far as to the moss clade. We carried out unbiased detection of SIRC ensembles in 10 plant genomes and found that, despite very high intraspecies heterogeneity, SIRC sets possess strong interspecies phylogenetic signal. Key messageShort Interrupted Repeats Cassettes are elements of ancient origin, and could potentially be used to trace organism history, and to facilitate syntheny and Hi-C analysis.

Accurate, simultaneous, and efficient quantification of chemically diverse phytohormone species is a critical task towards understanding the complex system of phytohormone signaling pathways. Quantification of phytohormones with the commonly used technique liquid chromatography coupled to tandem mass spectrometry is susceptible to the influence of non-phytohormone components present in the sample, a phenomenon referred to as matrix effect. To reduce matrix effect, some phytohormone quantification methods include additional steps of cleanup of crude extracts. However, to what extent additional purification steps provide increased accuracy compared to simpler, less laborious methods is seldomly evaluated. We evaluated three previously described phytohormone extraction methods, two of which include solid-phase extraction and one that does not, in their ability to minimize matrix effect and generate accurate estimates of phytohormone species spanning six classifications, from fruit and leaf tissue of Solanum lycopersicum cv. Micro-Tom (tomato). Our results show that, while the methods that included solid phase extraction occasionally outperformed each other regarding matrix effect and/or recovery efficiency for broad range of phytohormones, they rarely outperformed the simpler single-phase extraction method. Short AbstractAccurate, simultaneous quantification of chemically diverse phytohormones by LC-MS/MS is frequently confounded by matrix effects, leading to the incorporation of additional purification steps. We systematically compared three published extraction protocols with or without solid-phase extraction in tomato tissues across six hormone classes. Solid-phase methods occasionally improved matrix suppression or recovery, but did not consistently outperform the single-phase approach, questioning the added value of extra cleanup steps, particularly when high-throughput is desired, as in the case of systems biology interrogations.