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Seseli tomentosum Vis. is an endemic species distributed along the eastern coast of the Adriatic Sea. In this study, the leaf structure as observed by light and electron microscopy, the phytochemical composition of the volatile organic compounds, and the cytotoxic activity of S. tomentosum are presented. The secretory ducts located above and within the phloem and below the xylem part of the vascular bundle represent the first description of the leaf secretory structures of S. tomentosum. The essential oil and hydrosol were extracted from air-dried leaves by Clevenger distillation and analyzed by gas chromatography-mass spectrometry, combined with headspace solid-phase microextraction of volatiles from the hydrosol and fresh plant material. α-Amorphene, β-caryophyllene, germacrene D, β-cadinene, and α-copaene were the most abundant sesquiterpenes in the essential oil and fresh plant material. Among the monoterpenes, α-pinene was most abundant in the essential oil, limonene in fresh plant material, and α-terpineol in the hydrosol. Moderate cytotoxic activity of the methanolic extract of S. tomentosum, with higher inhibition of cell division observed in the human cervical cancer and osteosarcoma cell lines, and weaker activity in the healthy retinal pigmented epithelial and colon cancer cell lines, was detected using the MTS-based assay. With these results, we aim to highlight the potential of endemic plants, emphasizing the importance of studying species such as S. tomentosum and their contributions to biodiversity and human health as sources of bioactive compounds.

SnRK1 is an evolutionarily conserved protein kinase belonging to the SNF1/AMPK family of protein kinases that is central to adjusting growth in response to the energy status. Numerous studies have shown adaptive and developmental roles of SnRK1, but the understanding of the SnRK1 signaling network in monocots is limited. Using CRISPR/Cas9 mutagenesis to target the functional kinase subunits in rice, we carried out comprehensive phenotypic, transcriptomic, proteomic, and phosphoproteomic analyses of rice snrk1 mutants displaying growth defects under normal and starvation conditions. These analyses revealed the role of SnRK1 signaling in controlling growth and stress-related processes in both energy-sufficient and energy-limited conditions and pointed to the subfunctionalization of SnRK1 kinase subunit genes. In addition to the classical protein targets of SnRK1, phosphoproteomics revealed novel targets including the key components of intracellular membrane trafficking, ethylene signaling, and ion transport. The upregulation of stress-related processes and suppression of growth-related processes in snrk1 mutants correlated with their phenotypic defects. Overall, this study highlights a dual role of SnRK1 as a promoter of growth under favorable conditions and a critical regulator of adaptive response under stress conditions.

One class of enzymes that plant pathogens employ to manipulate innate immunity and physiology of the infected cells is host-targeted ADP-ribosyltransferases. The bacterial pathogen Pseudomonas syringae uses its Type III secretion system to inject several effector proteins with ADP-ribosyltransferase activity into plant cells. One of them, AvrRpm1, ADP-ribosylates the plasma membrane-associated RPM1-INTERACTING PROTEIN 4 (RIN4) in Glycine max and Arabidopsis thaliana to attenuate targeted secretion of defense-promoting compounds. Substrate identification of host-targeted ADP-ribosyltransferases is complicated by the biochemical lability of the protein modification during plant protein extraction and in several cases requires prior knowledge of plant immune signaling pathways that are impaired by the ADP-ribosylating Type III effector. Using the AvrRpm1-RIN4 pair as a proof of concept, we present an untargeted proteomics workflow for enrichment and detection of ADP-ribosylated proteins and peptides from plant cell extracts that in several cases provides site resolution for the modification.

The reproductive cycle of interior lodgepole pine spans approximately 26 months, with seed cones initiating in late summer, followed by pollen conelets emerging the next spring when pollination occurs. Fertilization and cone maturation occur in the subsequent spring and fall. Successful pollination, critical for seed conelet retention, requires an ~80% success rate. In June 2020, 30 pine clones (genotypes), with contrasting conelet abortion rates, were selected from a production seed orchard in Alberta, Canada. Sixty conelets per clone (30 aborted and 30 healthy) were collected for synchrotron radiation-based microcomputed tomography (SR-μCT) analysis. A subset of two contrasting clones (1401 and 596) was selected for detailed study. Conelets were transported to the Canadian Light Source in Saskatoon, Saskatchewan, for SR-μCT Phase Contrast Imaging (PCI), where samples were mounted and scanned using a PCO Edge 5.5 sCMOS camera and analyzed using Avizo software to visualize internal structures. Results showed that healthy conelets in clones 1401 and 596 had greater tissue volume (248.8 and 251.9 mm³, respectively) and more organized cavity spaces than aborted conelets (64.7 and 102.1 mm³, respectively), with differential internal air space volume (clone 1401: 8.7 mm³ in aborted vs. 17.5 mm³ in healthy; clone 596: 6.9 mm³ in aborted vs. 11.5 mm³ in healthy). Overall, SR-μCT imaging demonstrated significant advantages over traditional methods of visualization by providing a nondestructive, high-resolution analysis of internal structures. In addition, this technology enhances our understanding of pine reproductive processes, improving management practices in seed orchards-the delivery system for tree improvement programs.

This beginner's guide is intended for plant biologists new to network analysis. Here, we introduce key concepts and resources for researchers interested in incorporating network analysis into research, either as a stand-alone component for generating hypotheses or as a framework for examining and visualizing experimental results. Network analysis provides a powerful tool to predict gene functions. Advances in and reduced costs for systems biology techniques, such as genomics, transcriptomics, and proteomics, have generated abundant omics data for plants; however, the functional annotation of plant genes lags. Therefore, predictions from network analysis can be a starting point to annotate genes and ultimately elucidate genotype-phenotype relationships. In this paper, we introduce networks and compare network-building resources available for plant biologists, including databases and software for network analysis. We then compare four databases available for plant biologists in more detail: AraNet, GeneMANIA, ATTED-II, and STRING. AraNet and GeneMANIA are functional association networks, ATTED-II is a gene coexpression database, and STRING is a protein-protein interaction database. AraNet and ATTED-II are plant-specific databases that can analyze multiple plant species, whereas GeneMANIA builds networks for Arabidopsis thaliana and nonplant species and STRING for multiple species. Finally, we compare the performance of the four databases in predicting known and probable gene functions of the A. thaliana Nuclear Factor-Y (NF-Y) genes. We conclude that plant biologists have an invaluable resource in these databases and discuss how users can decide which type of database to use depending on their research question.

[This corrects the article DOI: 10.1002/pld3.70081.].

RNA editing and maturation are critical regulatory mechanisms in plant organelles, yet their quantification remains technically challenging. Traditional Sanger sequencing lacks sensitivity and reproducibility, whereas advanced next-generation sequencing (NGS) approaches, such as rRNA-depleted long non-coding (lnc) RNA-seq or targeted amplicon-seq, involve high costs, complex workflows, and limited accessibility. To address these limitations, I developed a rapid and cost-effective long-read sequencing approach, termed Target-Indexed-PCR (TIP) sequencing, for digital quantification of RNA editing and intron retention events in targeted chloroplast transcripts. This method combines multiplexed high-fidelity PCR amplification with Oxford Nanopore sequencing and custom in-house Perl and Python scripts for streamlined data processing, including barcode-based demultiplexing, strand reorientation, alignment to a pseudo-genome, manual editing-site inspection, and splicing variant identification. As a proof of concept, TIP sequencing was applied to ndhB and ndhD transcripts, two chloroplast NAD(P)H dehydrogenase genes with the highest number of known editing sites in Arabidopsis thaliana. These transcripts were analyzed both in an inducible CRISPR interference (iCRISPRi) system targeting MORF2, a key RNA-editing factor, and in MORF2-YFP transgenic lines with either overexpression or co-suppression silencing. My findings revealed dose- and development-dependent impacts of MORF2 on C-to-U editing efficiency. Moreover, I identified an accumulation of intron-retaining ndhB transcripts, specifically in Dex-treated iCRISPRi lines and in both MORF2-YFP overexpression and silencing rosette leaves, indicating impaired chloroplast splicing functions when MORF2 expression is perturbed beyond an as-yet-undefined threshold. The platform achieves single-molecule resolution, robust reproducibility, and high read coverage across biological replicates at a fraction of the cost of lncRNA-seq. Collectively, this study establishes TIP sequencing as a versatile, scalable, and affordable tool for targeted post-transcriptional analysis in plant organelles and expands our understanding of MORF2's role in chloroplast RNA maturation. By overcoming key limitations of existing approaches, TIP sequencing enables routine, site-specific quantification of post-transcriptional regulation in organelles, including RNA editing and splicing, making it broadly accessible to researchers studying plastid biology, stress responses, and organelle-nucleus communication.

Programmed cell death (PCD) mediates plant development and environmental interactions. Photosynthesis-derived singlet oxygen (¹O₂) is one of key reactive oxygen species (ROS) implicated in acclimation and PCD responses to environmental stress conditions. Using Arabidopsis thaliana cell suspension culture system, we characterized the PCD induced by Rose Bengal (RB), a photosensitizer generating ¹O₂ upon light exposure. Obtained results reiterated that RB-induced PCD is light and chloroplast dependent. Further, we demonstrate that PCD induced by RB involves calcium signaling and mitochondria, thus sharing common features with other forms of regulated cell death in plants. The PCD induced by RB was associated with early transcriptional reprogramming, involving switching off the primary metabolism and activation of stress response and cell death related pathways (e.g., oxidative stress, hypoxia, immunity, and salicylic acid). The constructed gene regulatory network featured ¹O₂-responsive genes and suggested involvement of transcription factor ANAC102 in retrograde regulation of RB-induced PCD. Interestingly, treatment with RB also induced light independent toxicity, showing features of uncontrolled, necrotic cell death. Presented findings highlight RB as a valuable tool for studying ¹O₂-induced PCD that may advance future work on chloroplast-mediated oxidative stress responses and enhancing plant resilience to climate change-related stressors through targeted modulation of ROS pathways.

Powdery mildew (PM), mainly caused by Podosphaera xanthii, is a severe destructive disease that threatens the production of cucurbit crops globally. Heterologous transformation has shown that the CmSGT1 gene (suppressor of the G2 allele of skp1) improved PM resistance in tobacco. However, the function of the gene in pumpkins (Cucurbita moschata) is largely unknown. Herein, transient CmSGT1 overexpression in pumpkin cotyledons inhibited the spore germination and mycelia growth of P. xanthii by inducing an increase in salicylic acid (SA) content, and exogenous SA intensified the inhibitory effect of the gene on the growth of P. xanthii. The β-glucuronidase activity of cotyledons transformed with the CmSGT1 promoter was induced by PM and signaling molecules (gibberellic acid, ethephon, SA, abscisic acid, and methyl jasmonate). The yeast one-hybrid assay verified that transcription factor CmWRKY21, CmWRKY31, and CmWRKY75 proteins interact with the CmSGT1 promoter. Transactivation analysis revealed that CmWRKY21 and CmWRKY31 significantly triggered the expression of GUS driven by the CmSGT1 promoter under PM. Furthermore, transient co-overexpression of CmWRKY21/CmWRKY31 and CmSGT1 enhanced the inhibitory effect on the growth of P. xanthii. In conclusion, the CmSGT1 gene is a PM resistance gene in pumpkin and is transcriptionally regulated by CmWRKY21 and CmWRKY31. Our study provides a reference for resistance breeding of pumpkins.

Flowering dogwood (Cornus florida L.) and Asian dogwood (Cornus kousa F. Buerger ex Hance) are popular deciduous ornamental trees native to a wide range of the eastern and southeastern United States and East Asia, respectively. Anthocyanin pigments enhance desirable pink or dark red colored bracts in dogwoods. Although anthocyanin biosynthesis is one of the best-studied biological processes in nature, genomic and genetic resources to understand the molecular regulation of its synthesis in dogwoods are still lacking. Two classes of genes control anthocyanin production; both structural genes and MYB transcription factors may function as positive or negative regulators of anthocyanin biosynthesis. To reveal the molecular mechanisms that govern color production in ornamental dogwoods, mature bracts of three cultivars of C. florida (white bracts: "Cloud Nine"; red bracts: "Cherokee Brave," and "Cherokee Chief") and two cultivars of C. kousa (light green bracts: "Greensleeves" and midtone pink bracts "Rosy Teacups") were sampled when color was maximally visible. Differential gene expression analysis of the RNAseq data identified 1156 differentially expressed genes in C. florida and 1396 in C. kousa. Phylogenetic analysis with functional orthologues in other plants grouped the candidate R2R3-MYB identified in this study into two distinct subgroups. CfMYB2, CfMYB3, and CkMYB2 belong to Subgroup 4, whereas CfMYB1 80 and CkMYB1 clustered in Subgroup 5. Genes in the former group repress anthocyanin and proanthocyanidin synthesis in flowering and Asian dogwoods, whereas genes in the latter increase it. Our study contributes to understanding processes behind anthocyanin production and lays the foundation for the future development of molecular markers for faster development of desirable red-bracted dogwoods.