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PAT: An Image Analysis Tool for Automated Scoring of Pollen in Alexander-Stained Anthers

Quantitative pollen viability analysis is a critical but labor-intensive step in plant reproductive biology. Existing deep-learning Segment Anything Models (SAM) fail to reliably segment viable pollen in Alexander-stained anthers. To address this, we fine-tuned an existing Cellpose-SAM model for pollen segmentation. We integrated it into PAT (Pollen Analysis Tool), a cross-platform desktop application. PAT features instance segmentation with interactive quality control, an in-app model retraining module, and publication-ready statistical outputs. We deployed PAT in an EMS suppressor screen of semi-sterile Arabidopsis smg7-6 mutants, enabling efficient candidate prioritization for whole genome sequencing and mapping candidate mutation. This screen led to the identification of a point mutation in CAP-D2 (capd2-2), a Condensin I subunit, that rescues the smg7-6 meiotic phenotype. Notably, mutation in a Condensin II subunits (CAP-D3 and CAP-H2) does not confer rescue. Further characterization suggests the capd2-2 allele is hypomorphic, showing no defects in vegetative growth, chromocenter compaction, or transposable element silencing. Collectively, we demonstrate that accessible AI tools have the potential to bridge gaps in plant phenotyping and accelerate the pace of biological discovery. HighlightWe combined AI-powered image analysis with an easy-to-use desktop app to automate plant pollen counting, then used it to identify a new genetic suppressor of meiotic defects.

plant biology↗

Variability in Fruit Yield and Quality of Genetically Diverse Tomato Cultivars in Response to Different Biochars

Background Intensive agricultural practices have reduced soil health thereby negatively impacting crop yields. There is a need to maintain healthy soils and restore marginal lands to ensure efficient food production. Biochar, a porous carbon-rich material generated from pyrolysis of various feedstock sources is receiving attention as a soil amendment that has the potential to restore soil organic carbon content and also enhance crop yields. However, the physical and chemical properties of biochar are influenced by pyrolysis parameters. These in turn determine its interaction with the soil, thereby influencing its biological properties in terms of impact on soil microcosm and plant productivity. While most studies report the evaluation of one biochar and a single plant cultivar, the role of the plant’s genetic background in responding to biochar as a soil amendment remains unanswered. The impact of six distinct biochars on agronomic performance and fruit quality of three genetically diverse tomato (Solanum lycopersicum) cultivars was evaluated to test the hypotheses that 1) biochars derived from different feedstock sources would produce unique phenotypes in a single cultivar of tomato, and 2) single feedstock-derived BC would produce different phenotypes in each of the three tomato cultivars.Results Different biochars impacted shoot dry weight, total fruit weight, and yield per plant in each cultivar differently. Both positive and negative effects were observed depending on the biochar-cultivar combination. In ‘Oregon Spring’, Ryegrass straw and CoolTerra biochar enhanced yield. In ‘Heinz’, an increase in fruit weight and citric acid was observed with several of the biochars. In ‘Cobra’, improved yields were accompanied by reduction in fruit quality parameters. Both hypotheses were supported by the data.Conclusions This study demonstrated that the genetic background of a plant is an important variable in determining the outcome of using biochar as a soil amendment. Strategies for application of biochar in agricultural production should consider the variables of soil type, feedstock source, pyrolysis parameters and plant genetic background for enhancing crop productivity and carbon sequestration.Competing Interest StatementAD serves as a consultant for AgEnergy Solutions, a biochar production startup company based in Spokane, WA, USA. AgEnergy Solutions had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.AbbreviationsBCBiocharSOMsoil organic matterSOCsoil organic carbonCTCool Terra®RGSRyegrass strawRGTRyegrass tailingsRTRussian thistleTMPThermomechanical pulpWWalnutSEMScanning Electron MicroscopyEXenergy-dispersive X-ray spectroscopy – EDXw/wweight/weightHigh Pressure SodiumHPStotal soluble solidsTSSNnitrogenPphosphorusKpotassiumCacalciumSsulfurMgmagnesiumMomolybdenumSisiliconClchlorineNasodiumAlaluminumView Full Text

plant biology↗

User-friendly electron microscopy protocols for the visualization of biological macromolecular complexes in three dimensions: Visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic 3-dimensional (3D) parts. Therefore, it is critical to study biological structures in 3D and at high resolutions to gain insights into their physiological functions. Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution. However, many of these protocols require specialized equipment and personnel to complete them. Here we present novel accessible protocols to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolutions, focusing on Arabidopsis clathrin-coated vesicles (CCVs) - an essential trafficking organelle lacking detailed structural characterization due to their low preservation in classical electron microscopy techniques. First, we establish a protocol to visualize CCVs in unroofed cells using scanning-transmission electron microscopy (STEM) tomography, providing sufficient resolution to define the clathrin coat arrangements. Critically, the samples are prepared directly on electron microscopy grids, removing the requirement to use extremely corrosive acids, thereby enabling the use of this protocol in any electron microscopy lab. Secondly, we demonstrate this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells. Finally, to facilitate the high-throughput and robust screening of metal replicated samples, we provide a deep learning analysis workflow to screen the pseudo 3D morphology of CCVs imaged with 2D modalities. Overall, we present accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

plant biology↗

Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in Kumquat

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants. In this study, we demonstrated that cold-induced expression of protease inhibitor FmASP in a citrus relative species kumquat (Fortunella margarita (Lour.) Swingle) contributes to its freezing tolerance by regulating protein degradation. First, we found that only cold-acclimated kumquat plants, although with extensive leaf cellular damage during freezing, are able to resume their normal growth upon stress relief. To dissect the impact of cold acclimation on this extraordinary freezing tolerance, we performed protein abundance assay and quantitative proteomics analysis of kumquat leaves subjected to cold acclimation (4 {degrees}C), freezing treatment (-10 {degrees}C) and post-stress recovery (25 {degrees}C). FmASP and a few non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of freezing treatment. FmASP was further characterized as a robust inhibitor that inhibits the degradation capacity of multiple proteases. In addition, heterogeneous expression of FmASP in Arabidopsis confirmed its positive function in freezing tolerance. Finally, we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation and consequently maintains essential cellular function for freezing stress recovery. These findings revealed the important role of protease inhibition on freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance. One-sentence summaryA protease inhibitor of Fortunella margarita enhances protein stability and freezing tolerance by regulating non-specific protease degradation

plant biology↗

HalluCodon enables species-specific codon optimization using multimodal language models

Codon optimization is widely used in transgenic crop development, plant synthetic biology, and molecular farming to improve heterologous protein expression in plant cells. Increasing availability of plant omics data now enables optimization strategies that account for species-specific sequence features. We developed HalluCodon, a customizable framework that uses multimodal language models to design coding sequences tailored to individual plant species. The framework allows users to fine tune pre-trained protein and RNA language models with their own datasets to build species-specific codon optimization models. The current implementation includes base models trained on coding sequences and proteomes from fifteen plant species. HalluCodon generates coding sequences through a hallucination-based design strategy guided by two predictive modules that evaluate coding sequence naturalness (CodonNAT) and expression potential (CodonEXP). Benchmark tests using representative proteins show that the generated sequences reproduce host-specific codon usage patterns and support high expression levels in plant systems.

bioinformatics↗

Genome-wide identification and characterization of DNA/RNA differences associated with Fusarium graminearum infection in wheat

RNA editing (DNA/RNA differences) as a post-transcriptional modification approach to enrich genetic information, plays the crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genome, RNA editing in plant nuclear genome, especially those associated with Fusarium head blight (FHB), is not well studied at present. Here, we investigated the DNA/RNA differences associated with FHB through a novel method by comparing the RNA-seq data from Fusarium-infected and control samples from 4 wheat genotypes. A total of 187 DNA/RNA differences were identified in 36 wheat genes, representing the first landscape of the FHB-responsive RNA editome in wheat. Furthermore, all of these 36 edited genes were located in the FHB related co-expression gene modules, which may involve in regulating FHB response. Finally, the effects of DNA/RNA differences were systematically investigated to show that they could cause the change of RNA structure and protein structure in edited genes. In particular, the G to C editing (chr3A_487854715) in TraesCS3A02G263900, which is the orthology of OsRACK1, resulted that it was targeted by tae-miR9664-3p to control its expression in different genotype through different editing efficiency, suggesting RNA editing could mediate miRNA to participate in the regulation network of FHB tolerance. This study reported the first wheat DNA/RNA differences associated with FHB, which not only contribute to better understand the molecular basis underlying FHB tolerance, but also shed light on improving FHB tolerance through epigenetic method in wheat and beyond.

plant biology↗

Core circadian clock and light signalling genes brought into genetic linkage across the green lineage

The circadian clock ensures that biological processes are phased to the correct time of day. In plants the circadian clock is conserved at both the level of transcriptional networks as well as core genes. In the model plant Arabidopsis thaliana, the core circadian singleMYB (sMYB) genes CCA1 and RVE4 are in genetic linkage with the PSEUDO-RESPONSE REGULATOR (PRR) genes PRR9 and PRR7 respectively. Leveraging chromosome-resolved plant genomes and syntenic ortholog analysis it was possible to trace this genetic linkage back to the basal angiosperm Amborella and identify an additional evolutionarily conserved genetic linkage between PIF3 and PHYA. The LHY/CCA1-PRR5/9, RVE4/8-PRR3/7 and PIF3-PHYA genetic linkages emerged in the bryophyte lineage and progressively moved within several genes of each other across an array of higher plant families representing distinct whole genome duplication and fractionation events. Soybean maintains all but two genetic linkages, and expression analysis revealed the PIF3-PHYA linkage overlapping with the E4 maturity group locus was the only pair to robustly cycle with an evening phase in contrast to the sMYB-PRR morning and midday phase. While most monocots maintain the genetic linkages, they have been lost in the economically important grasses (Poaceae) such as maize where the genes have been fractionated to separate chromosomes and presence/absence variation results in the segregation of PRR7 paralogs across heterotic groups. The evolutionary conservation of the genetic linkage as well as its loss in the grasses provides new insight in the plant circadian clock, which has been a critical target of breeding and domestication. Summary SentenceThe genetic linkage of the core circadian clock components has evolutionary origins in bryophytes and sheds light on the current functioning and selection on the circadian clock in crops.

plant biology↗

Genome-scale mRNA transcriptomic insights into the mature growth dynamic of Cigar tobacco leaves

The harvest time is a key factor for cigar leaves with high quality, which varies greatly depending on the environment. Here, we performed a genome-scale mRNA transcriptomic analysis on the cigar cultivar CX-26 (Nicotiana tabacum L.) to evaluate the relationship between gene expression and growth state. The leaves were harvested with 67 (T1), 71(T2) and 75 (T3)-day growth. A total of 80,502 genes were detected in the CX-26 leaves, of which 64,611 genes were annotated on the reference genome. Principal component analysis showed that T1 and T2 leaves had a high overlapping pattern, while T3 leaves were distinct. Indeed, T1 and T2 leaves had fewer differential expressing genes (DEGs), while T3 leaves had 26,456 DGEs from T2 leaves, supporting the distinct growth of T3 leaves. GO annotations mainly enriched the photosynthesis-related metabolic process, catalytic activity and binding biological processes. KEEG analysis identified the key pathways including photosynthesisantenna proteins, plant hormone/MAPK signaling pathway and plant-pathogen interaction. The maturity regulation and defense response-associated hormones abscisic acid and jasmonate acid were higher in the T3 leaves than that in T1 and T2 leaves confirming the KEEG analysis. Furthermore, several photosynthesis-related enzymes and a transcription factor were highlighted in the gene regulatory network, which might regulate the dynamics of carbohydrate metabolism, lipid metabolism and energy metabolism. In summary, our study provides insight into the growth state of CX-26 cigar leaves.

plant biology↗

Diverse photoperiodic gene expression patterns are likely mediated by distinct transcriptional systems in Arabidopsis

Photoperiod is an annual cue measured by biological systems to align growth and reproduction with the seasons. In plants, photoperiodic flowering has been intensively studied for over 100 years, but we lack a complete picture of the transcriptional networks and cellular processes that are photoperiodic. We performed a transcriptomics experiment on Arabidopsis plants grown in 3 different photoperiods, and find that nearly one-third of the known genes show photoperiodic alteration in gene expression. Gene clustering, daily expression integral calculations and cis-element analysis then separate photoperiodic genes into co-expression subgroups that display 19 diverse seasonal expression patterns, opening the possibility that many photoperiod measurement systems work in parallel in Arabidopsis. Then, functional enrichment analysis predicts co-expression of important cellular pathways. To test these predictions, we generated a comprehensive catalog of genes in the phenylpropanoid biosynthesis pathway, overlaid gene expression data and demonstrated that photoperiod intersects with the two major phenylpropanoid pathways differentially, controlling flavonoids but not lignin. Finally, we describe the development of a new app that visualizes photoperiod transcriptomic data for the wider community.

plant biology↗

Homologous ABA-independent kinase tracks coalesced into osmotic stress circuits during plant terrestrialization

Land plants possess a unique system for responding to environmental stressors. How this system evolved during plant terrestrialization remains one of the major questions in plant evolutionary biology. To retrace this process, it is essential to study both land plants and their closest algal relatives, the zygnematophytes. Using the single-celled zygnematophyte Mesotaenium, we integrated physiological stress experiments with phosphoproteomics, genome-wide transcription factor binding analyses, and protein-protein interaction studies to investigate the architecture of a key stress response pathway: the signaling cascade homologous to the plant abscisic acid (ABA)-mediated pathway. Our results highlight the roles of histidine kinases (HKs) and calcium-dependent protein kinases (CDPKs) in osmotic stress signaling. Focusing on SnRK2 and ABF, key components at the downstream end of the canonical ABA signaling pathway, we provide evidence that ABF plays a central role in osmotic stress responses even in the absence of ABA. Together, our data reveal the coordinated action of parallel functional modules that were likely integrated into the ABA response cascade during plant terrestrialization.

evolutionary biology↗

Remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha

Gametogenesis is an essential biological event for sexual reproduction in various organisms. Bryophytes and some other plants employ motile sperms (spermatozoids) as male gametes, which self-locomote to the egg cells to accomplish fertilization. Spermatozoids of bryophytes harbor distinctive morphological characteristics, including the cell body with a helical slender shape and two motile flagella at the anterior edge. During transformation from a spermatid to spermatozoid (spermiogenesis), the shape and cellular contents of spermatids are dynamically reorganized. However, how each organelle is reorganized during plant spermiogenesis remains obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to cellular and nuclear shapes and flagella development. We then examined the remodeling of microtubules and reorganization of endomembrane organelles during spermiogenesis. The results indicate that the state of post-translational modification of tubulin is dynamically changed during the formation of the flagella and spline, and the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner during spermiogenesis. These findings are expected to provide useful indexes to classify developmental and subcellular processes of spermiogenesis in bryophytes. Summary statementWe classified developmental processes of spermatozoids into 1 + 5 stages and characterized remodeling of organelles and microtubules during spermiogenesis in the liverwort Marchantia polymorpha.

plant biology↗

Functional profiling of the male gametophyte-specific promoter fragment of Arabidopsis PIRL6 gene and prediction of cis-regulatory elements

Gametophyte-specific promoters drive the expression of genes in male and/or female gametophytes. These have applications in breeding experiments, gene function identification, developmental biology-related studies, and off-lately in genome editing also. The Plant Intracellular Ras-group Leucine-Rich-Repeat 6 (PIRL6) gene is known to be necessary for male and female gametogenesis. Using the GUS- based deletion analysis, we have identified the PIRL6 promoter length that is essential for exclusive expression in the male gametophyte of Arabidopsis thaliana. We studied the strength of various lengths of PIRL6 promoters in different tissues, by GUS expression quantification. The male-gametophyte-specific promoter segment (PA1) exhibited stronger expression in mature anthers than the younger ones. We identified 50 other genes that co-expressed with PIRL6 in Arabidopsis using the Expression Angler tool. Gene ontology (GO) analysis shows that these 51 co-expressing genes were predominantly involved in cell differentiation. By comparing the promoter sequences of these 51 genes, the presence of three over-represented known motifs, POLLEN1LELAT52, ACGTATERD1 and CIACADIANLELHC was identified. We could also predict the presence of three novel cis-regulatory elements in the co-expressing gene network using the MEME suite tool. Additionally, we confirmed the functionality of the ABRE and P-box elements present in PIRL6 promoter using the tobacco leaf transient assay. Thus, we cloned and functionally confirmed the promoter region of PIRL6 required for male gametophyte-specific expression, compared this promoter with those of 50 other co-expressed genes and predicted their functions, and analyzed their cis regulatory regions and predicted three novel motifs also. Key messageA pollen-specific promoter fragment of PIRL6 was functionally characterized using GUS-based deletion analysis. Fifty other co-expressed genes were compared and novel cis-regulatory elements were predicted. Two hormone-responsive elements in the PIRL6 promoter were found to influence the GUS expression. PA1 promoter can be used in experiments that require male gametophyte-specific expression of genes.

plant biology↗

Genome-wide identification of conserved RNA structure in plastids

Plastid genomes are deeply evolutionary conserved. RNA structures within the primary or mature transcript play central role in plastid regulation of RNA processing, stability, and translation. However, the identity and conservation of RNA structures selected in plastid's evolution are still largely elusive. Here, we developed a stringent, covariation-based pipeline that perform an unbiased screen for conserved RNA secondary structures across entire plastid genomes. We analysed ~14,000 plastid genomes and identified a repertoire of 57 high-confidence conserved structures. We recovered known functional classes, e.g., 16S rRNA, tRNA, group II intron, and 3' end stem-loop, evidencing that our genome-wide analysis is reliable. We further uncovered novel putative cis-acting structures within the UTRs and introns of key photosynthetic genes, including psbN, clpP, and atpF, as well as putative trans-acting antisense RNAs to petB and psbT, suggesting uncharacterized elements with major regulatory function. Experimental in vivo RNA probing demonstrated that nearly half of the conserved structures adopt the predicted conformation in Arabidopsis plastid. Our comprehensive, yet stringent atlas of conserved plastid RNA structures provides the foundations for new regulatory discoveries in plastid biology.

plant biology↗

Genome-wide characterization, evolutionary analysis of WRKY genes in Cucurbitaceae species and assessment of its roles in resisting to powdery mildew disease

The WRKY proteins constitute a large family of transcription factors that have been known to play a wide range of regulatory roles in multiple biological processes. Over the past few years, many reports have focused on analysis of evolution and biological function of WRKY genes at the whole genome level in different plant species. However, little information is known about WRKY genes in melon (Cucumis melo L.). In the present study, a total of 56 putative WRKY genes were identified in melon, which were randomly distributed on their respective chromosomes. A multiple sequence alignment and phylogenetic analysis using melon, cucumber and watermelon predicted WRKY domains indicated that melon WRKY proteins could be classified into three main groups (I-III). Our analysis indicated that no recent duplication events of WRKY genes were detected in melon, and strong purifying selection was observed among the 85 orthologous pairs of Cucurbitaceae species. Expression profiles of CmWRKY derived from RNA-seq data and quantitative RT-PCR (qRT-PCR) analyses showed distinct expression patterns in various tissues, and the expression of 16 CmWRKY were altered following powdery mildew infection in melon. Besides, we also found that a total of 24 WRKY genes were co-expressed with 11 VQ family genes in melon. Our comparative genomic analysis provides a foundation for future functional dissection and understanding the evolution of WRKY genes in cucurbitaceae species, and will promote powdery mildew resistance study in melon.

plant biology↗

Kinetic 13CO2 mapping revealed distinct light-dark metabolic transition phenotypes in Brassica napus seedlings under visible and UV-B light

Dynamic stable-isotope tracing using 13CO2 has gained significant attention in systems biology due to its potential to visualise carbon assimilation patterns. However, the plant metabolic phenotypes under visible and UV-B light, explaining the light-to-dark transition between photoperiods, remain unexplored. In this study, we investigated the dynamics of photosynthetic carbon assimilation and resource partitioning during the day-night transition under visible and UV-B light in Brassica napus seedlings. Kinetic 13CO2 tracing via the analysis of mass isotopomer distributions of metabolite fragments using GC-MS revealed reprogramming of the source-sink carbon dynamics. While visible light enabled the dynamic redistribution of newly fixed carbon during the light and dark photoperiod, de novo biosynthesis of shikimic acid, TCA cycle intermediates and metabolites of the glutamate-GABA metabolism was strongly favoured in dark metabolism. In contrast, a delayed and reduced photosynthetic carbon assimilation response was observed in the UV-B phenotype during the light period. Moreover, towards the late light period, de novo biosynthesis of sucrose, shikimic acid, phenylalanine, citric, succinic and malic acid was favoured, along with a greater reliance on pre-existing carbon pools for other metabolites. However, ketoglutarate, succinic acid, malic acid and GABA showed limited de novo synthesis in the dark period. Across both light regimes, amino acid pools largely remained in constant sync with the pre-existing pools during the light-dark transition. Overall, our findings demonstrate that light quality and photoperiod-driven metabolic transitions distinctly shape plant metabolic phenotypes.

plant biology↗

Correlates of hybridization in plants

Hybridization is a biological phenomenon increasingly recognized as an important evolutionary process in both plants and animals, as it is linked to speciation, radiation, extinction, range expansion and invasion, and allows for increased trait diversity in agricultural and horticultural systems. Estimates of hybridization frequency vary across taxonomic groups, and previous work has demonstrated that some plant groups hybridize more frequently than others. Here, we ask on a global scale whether hybridization is linked to any of 11 traits related to plant life history, reproduction, genetic predisposition, and environment or opportunity. Given that hybridization is not evenly distributed across the plant tree of life, we use phylogenetic generalized least squares regression models and phylogenetic path analysis to detect statistical associations between hybridization and plant traits at both the family and genus levels. We find that perenniality and woodiness are each associated with an increased frequency of hybridization in univariate analyses, but path analysis suggests that the direct linkage is between perenniality and increased hybridization (with woodiness having only an indirect relationship with hybridization via perenniality). Associations between higher rates of hybridization and higher outcrossing rates, abiotic pollination syndromes, vegetative reproductive modes, larger genomes, and less variable genome sizes are detectable in some cases but not others. We argue that correlational evidence at the global scale, such as that presented here, provides a robust framework for forming hypotheses to examine and test drivers of hybridization at a more mechanistic level.\n\nIMPACT SUMMARYAlthough historically thought of as rare, inter-specific mating is increasingly recognized as an important evolutionary process. Hybridization can generate increased genetic and morphological variation and has been tied to increased diversification and other biological phenomena such as geographic range expansion and the success of invasive species. Here, we examine hybridization of plants on a global scale. Previous work has demonstrated that some plant groups hybridize more than others, but the reasons for this pattern remain unclear. We combine data from eight regional floras with trait data to test for associations between hybridization and different aspects of plant biology, such as life history, growth form, reproduction, and opportunity, all while accounting for the fact that plant lineages are related to each other.\n\nWe find that plant groups that are dominated by perennial species and species with woody growth forms tend to hybridize more than those dominated by annual or herbaceous species. We also find some evidence that frequent hybridization is found in plant families that are predominantly pollinated abiotically (such as by wind or water) or have higher rates of outcrossing, plant genera that have less variable genome sizes, and plant groups (both genera and families) that can reproduce asexually and have larger genome sizes. This study provides the first analysis of the global correlates of hybridization in plants. Although this correlational evidence does not provide any mechanistic explanations for these patterns, the trends we find are novel in terms of both geographic and taxonomic sale. The correlations detected provide robust hypotheses for understanding the conditions for hybridization and its contributions to evolution.

evolutionary biology↗

Haplotype resolved DNA methylome of African cassava genome

Cytosine DNA methylation is involved in biological processes such as transposable element (TE) silencing, imprinting, and X chromosome inactivation. Plant methylation is mediated by MET1 (mammalian DNMT1), DRM2 (mammalian DNMT3), and two plant-specific DNA methyltransferases, CMT2 and CMT3 (Law and Jacobsen, 2010). De novo DNA methylation in plants is established by DRM2 via the plant specific RNA-directed DNA methylation (RdDM) pathway that depends on two DNA-dependent RNA polymerases, Pol IV and Pol V (Gallego-Bartolome et al., 2019; Law and Jacobsen, 2010; Stroud et al., 2013). The DNA methylome of cassava has been previously documented based on its haploid collapsed genome (Wang et al., 2015). Since the cassava genome is highly heterozygous, DNA methylome analysis of the haplotype-collapsed genome misses many features of the methylome. With the development of long read sequencing and chromosomal conformation capture techniques, haplotype resolved genomes are available for highly heterozygous genomes (Mansfeld et al., 2021; Qi et al., 2022; Zhou et al., 2020), which provides high-quality reference genomes facilitating studies of haplotype resolved DNA methylomes.

plant biology↗

Comparative Restriction Enzyme Analysis of Methylation (CREAM) Reveals Methylome Variability Within a Clonal In Vitro Cannabis Population

The primary focus of medicinal cannabis research is to ensure the stability of cannabis lines for consistent administration of chemically consistent products to patients. In recent years, tissue culture has emerged as a valuable technique for genetic preservation and rapid production of cannabis clones. However, there is concern that the physical and chemical conditions of the growing media can induce somaclonal variation, potentially impacting the viability and uniformity of clones. To address this concern, we developed Comparative Restriction Enzyme Analysis of Methylation (CREAM), a novel method to assess DNA methylation patterns and used it to assess a population of 78 cannabis clones maintained in tissue culture. Through bioinformatics analysis of the methylome, we successfully detected 2,272 polymorphic methylated regions among the clones. Remarkably, our results demonstrated that DNA methylation patterns were preserved across subcultures within the clonal population, allowing us to distinguish between two subsets of clonal lines used in this study. These findings significantly contribute to our understanding of the epigenetic variability within clonal lines in medicinal cannabis produced through tissue culture techniques. This knowledge is crucial for understanding the effects of tissue culture on DNA methylation and ensuring the consistency and reliability of medicinal cannabis products with therapeutic properties. Additionally, the CREAM method is a fast and affordable technology to get a first glimpse at methylation in a biological system. It offers a valuable tool for studying epigenetic variation in other plant species, thereby facilitating broader applications in plant biotechnology and crop improvement.

plant biology↗