Parkinson biomarker determination with an Au microflower-enhanced electrochemical immunosensor using non-Faradaic capacitance measurements.

This work comprehends the development and characterization of a carbon black-based electrode modified with Au microflowers to increase its effect as a capacitance biosensor for the determination of PARK7/DJ-1. Due to its high surface-to-volume ratio and biocompatibility, Au particles are suitable for antibody binding, and by monitoring surface capacitance, it is possible to identify the immune-pair interaction. Au microflowers allowed the adequate immobilization of Parkinsonian-related proteins: PARK7/DJ-1 and its antibody. The protein is associated with several antioxidant mechanisms, but its abnormal concentrations or mutations can be the cause of the loss of dopaminergic neurons, leading to Parkinson's disease. The device was characterized by scanning electron microscopy and cyclic voltammetry, revealing the flower-like structures and the electrochemically-interest enhancements they provide, such as increased heterogeneous electron transfer rate coefficient and electroactive area. The self-assembled monolayers of different molecules were optimized with the aid of 22 central composite experiments and a linear calibration curve was obtained between 0.700 and 120 ng mL-1 of PARK7/DJ-1, with a limit of detection of 0.207 ng mL-1. The data confirms that the addition of Au microflowers enhanced the electrochemical signal of the device, as well as allowed for the determination of an early stage Parkinson's disease biomarker with appreciable analytical performance.

Natural coil springs: biomechanics and morphology of the coiled tendrils of the climbing passion flower Passiflora discophora.

Tendrils of climbing plants possess a striking spring-like structure characterized by a minimum of two helices of opposite handedness connected by a perversion. By performing tensile experiments and morphological measurements on tendrils of the climbing passion flower Passiflora discophora, we show that these tendril springs act as coil springs within the plant's attachment system and resemble technical coil springs. However, tendril springs have a low spring index and a high pitch angle compared with typical metal coil springs resulting in a more complex loading situation in the plant tendrils. Moreover, the tendrils undergo a drastic shift from the fresh turgescent stage to a dried-off and dead senescent stage. This entails changes in material properties (elastic modulus in tension), morphology (tendril and helix diameter, number of windings), anatomy (tissue composition), and failure behavior (susceptibility to delamination) and reduces the degree of elasticity and strain at failure of the tendrils. Nevertheless, senescent tendrils remain functional as springs and maintain high energy dissipation capacity and high break force. This renders the system highly energy efficient, as the plant no longer needs to metabolically sustain the died-back tendrils. Because of its energy-storing spring system, its high energy dissipation and high safety factor, the attachment system can be considered a 'fail-safe' system. STATEMENT OF SIGNIFICANCE: : The use of coil springs as mechanical devices is not restricted to man-made machinery; striking spring structures can also be found within the attachment systems of climbing plants. Passiflora discophora climbs by using long thin tendrils with adhesive pads at their tips. Once the pads have attached to a support, the tendrils coil and form a spring-like structure. Here, we analyze the form and mechanics of these 'tendril springs', compare them with conventional technical coil springs, and discuss changes in the tendril springs during plant development. We reveal the main features of the attachment system, which might inspire new artificial attachment devices within the emerging field of plant-inspired soft-robotics.

Phytochemical Analysis and Bioactive Properties of Opuntia dillenii Flower Extracts, Compound, and Essential Oil.

New research is exploring the enhanced efficacy of antioxidant and antimicrobial compounds developed from Opuntia dillenii flowers, a multifaceted source with pharmacological effects such as antioxidant and microbicide activity indexes showing diverse medical capabilities. The purpose of this study was to determine the chemical composition, isolate the active compounds, and evaluate their antioxidant properties as well as antibacterial potential through HPLC-MS in flower extract from Opuntia dillenii. The extracts were analyzed by high-performance liquid chromatography (HPLC), and essential oil compounds were identified by gas chromatography (GC). Antioxidant properties were assessed using DPPH and ferric-reducing power (FRAP) assays. Antibacterial potential was evaluated using disk diffusion and microdilution methods. Nutritional studies of the flower indicated that it contained moderate levels of sugars (4.27% ± 0.240), proteins (1.913% ± 0.268), and microelements (potassium as a major element), sodium, and calcium, with concentration values of 2.267%, 0.55%, and 0.424%, respectively. Total phenolic content ranged from 1.61 ± 0.37 mg GAE/g (hexane extract) to 34.45 ± 0.42 mg GAE/g (ethanol extract). The study highlighted the richness of secondary metabolites, such as methylated flavonoids (quercetin 3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside), and identified essential oil compounds like trimethylsilyl hexadecenoate, squalene, gamma-eudesmol, and citronellol. Antioxidant activities revealed stronger activity in the butanolic extract, while isorhamnetin-3-O-rutinoside exhibited moderate antioxidant effects. These results provide the rationale for the potential incorporation of Opuntia dillenii flower extracts in food, cosmetics, and pharmaceutical products as a sustainable natural alternative with broad implications for human health.

Comparative Component Composition of Flowers in Several Syringa Species (Oleaceae).

Because insufficient relevant data are available, the component composition of flowers was studied comparatively in six lilac species of the genus Syringa L. (Oleaceae): S. emodi Wall., S. josikaea Jacg., S. komarowii Schneid., S. reticulata (Blume) H. Hara ssp. amurensis (Rupr.) P. S. Green et M. C. Chang, S. sweginzowii Koehne et Lingelsh, and S. vulgaris L. Plants were from the collection of the South Ural Botanical Garden-Institute. Contents were established for major groups of biologically active substances (BASs): flavonoids, hydroxycinnamic acids, tannins, and organic acids. The highest contents of all BAS groups were observed in S. komarowii flowers: flavonoids, 4.25 ± 0.21%; hydroxycinnamic acids, 6.13 ± 0.29%; tannins, 9.3 ± 0.5%; ascorbic acid, 0.468 ± 0.022%; and organic acids, 10.7 ± 0.5%. The contents were somewhat lower in S. emodi and S. sweginzowii and minimum in S. josikaea. Comparisons of the qualitative and quantitative BAS profiles showed that certain Syringa species cultivated in the Republic of Bashkortostan are promising to further study in terms of their possible use in scientific medicine and the development of herbal remedies.

How pollen and ovule development underlay dioecy in Chloroluma gonocarpa (Sapotaceae).

Previous studies have determined that Chloroluma gonocarpa (Sapotaceae), is a species that has cryptic dioecy. This type of sexual system is characterized by flowers that are morphologically perfect (both sexual whorls are present) but functionally pistillate or staminate (in each type of flower one of the sexual whorls is non-functional). In C. gonocarpa the pistillate flowers present well-developed stigma, functional ovules, and staminodes, while the staminate flowers present a poorly developed stigma, collapsed ovules, and pollen-producing anthers. In angiosperms, the abortion of sexual organs can occur at different stages of development (from pre-meiosis to post-meiosis), that is why we conducted an anatomical analysis of both flower types at various developmental stages. Using light microscopy, we described the processes of sporogenesis and gametogenesis to establish when the staminate flowers lose their pistillate function. To achieve this, we collected, fixed, and processed the flowers following conventional anatomical techniques for observation under a light microscope. Our findings reveal that pollen development occurs only in staminate flowers, while ovule development begins in both types of flowers but ceases in staminate flowers due to post-meiosis abortion. In contrast, normal development continues in pistillate flowers. These results suggest that dioecy in C. gonocarpa may have arisen from a gynodioecious pathway.

Beyond the Scent: New Evidence about Micromorphological, Phytochemical and Biological Features of Plumeria rubra 'Tonda Palermitana' (Apocynaceae).

Plumeria rubra L. is an ornamental Caribbean plant widely known for its ethnobotanical uses and pharmacological activities. The 'Tonda Palermitana' cultivar, on which no data are to date available, is commonly cultivated in Sicily. The aim of our study was to characterize the micro-morphological features of leaves and flowers of this cultivar by light and Scanning Electron Microscopy and to investigate the phytochemical profile and the biological properties of their food-grade extracts (LE and FE, respectively) by LC-DAD-ESI-MS analysis and different in vitro assays. Numerous branched laticifers were observed, and their secretion contained alkaloids and lipophilic compounds as confirmed by histological analyses. Phytochemical analyses showed the presence of alkaloids (9%), terpenoids (13%) and fatty acids (6%), together with a very abundant presence of iridoids (28%) and polyphenols (39%). The most notable biological activity of both extracts appears to be the antioxidant one, showing half-inhibitory concentrations (IC50) about 5 times lower than those detected in anti-inflammatory assays (383.74 ± 5.65 and 232.05 ± 2.87 vs. 1981.23 ± 12.82 and 1215.13 ± 10.15, for FE and LE, respectively), with LE showing the best, and statistically significant (p < 0.001), biological activity. These results allow us to speculate promising nutraceutical and cosmeceutical applications for this old Sicilian cultivar.

Online identification of potential antioxidant components and evaluation of DNA oxidative damage protection ability in Prunus persica flowers.

A high performance liquid chromatography-ultraviolet-visible detector-electrospray ionization-ion trap-time-of-flight-mass spectrometry-total antioxidant capacity determination (HPLC-UVD-ESI-IT-TOF-MS-TACD) new online technique was developed for efficient screening of potential antioxidant active components in Prunus persica flowers (PPF) from 4 origins. Through this online system, 46 compounds were initially identified, while 20 compounds with DPPH binding activity and 21 compounds with FRAP binding activity were detected. The antioxidant activities of 9 compounds obtained from the screening were then validated in DNA oxidative damage protection study. The results showed that this online system can cope well with the complexity of the samples. This also provides technical basis for rapid screening of antioxidant resources of PPF. In short, this study made the chemical composition of PPF more abundant and its potential antioxidant active compounds more explicit, which provided new ideas for the detection and development of natural antioxidants and provided scientific basis for PPF as functional food.

Genome-Wide Analyses of CCHC Family Genes and Their Expression Profiles under Drought Stress in Rose (Rosa chinensis).

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

Evaluating the Performance of Cocopeat and Volcanic Tuff in Soilless Cultivation of Roses.

Roses are increasingly being grown in soilless systems to increase productivity and reduce the challenges associated with soil-based cultivation. This study investigates the effects of using cocopeat and volcanic tuff substrates, the particle size of tuff, and substrate depth on the growth and flower quality of roses (Rosa hybrida L. cv. top secret) grown under greenhouse conditions. The treatments were cocopeat, tuff, cocopeat-tuff mixture, and tuff of particle size of 2 to 4 mm, 0 to 4 mm, and 0 to 8 mm at depths of 20 cm and 40 cm. The results showed that cocopeat had the highest water-holding capacity and photosynthetic rate. Tuff substrates had higher chlorophyll content throughout the growing season. Although flower numbers per plant in cocopeat and tuff from 0 to 8 mm at a depth of 20 cm were statistically similar, tuff from 0 to 8 mm had longer flowering stems and larger post-harvest flower diameters than cocopeat. An increase in the tuff depth from 20 to 40 cm decreased the flower number and main stem diameter. In conclusion, while cocopeat promotes rapid initial growth, volcanic tuff substrates, particularly tuff from 0 to 8 mm at a depth of 20 cm, provide long-term benefits for flower quality and plant health.

Comparative Study of Flavonoid Profiles, Antioxidant, and Antiproliferative Activities in Hot-Air and Vacuum Drying of Different Parts of Pitaya (Hylocereus undatus Britt) Flowers.

Pitaya flower, a medicinal and edible plant commonly used in tropical and subtropical regions, was the focus of this study, which compared the effects of hot-air drying (HAD) and vacuum drying (VD) on phytochemical profiles and biological activities of its four parts: calyx, petals, stamens, and pistils. Both drying methods significantly increased the total phenolic content (TPC) of pitaya flowers, with values ranging from 1.86 to 3.24 times higher than those of fresh samples. Twelve flavonoid compounds were identified in pitaya flowers, with the glycoside derivatives of three flavonols (kaempferol, isorhamnetin, and quercetin) being the most abundant. VD resulted in 1.15 times higher total flavonoid glycoside content than HAD, whereas in petals, HAD yielded a total flavonoid glycoside content 1.21 times higher than VD. Both HAD and VD effectively increased the antioxidant capacities of pitaya flowers, though the difference between the two methods was not significant. Additionally, both drying methods enhanced the antiproliferative activity of pitaya flowers, with HAD showing a more significant effect than VD. The present study emphasized the efficacy of drying methods for enhancing flavonoids in pitaya flowers and provided insights for functional products' innovation with different parts of pitaya flowers.

Constituents from Dolichos lablab L. Flowers and Their Anti-Inflammatory Effects via Inhibition of IL-1ß Release.

The occurrence of inflammation is closely related to the activation of the NLRP3 inflammasome. IL-1ß produced during the activation of the NLRP3 inflammasome has strong pro-inflammatory activity and can also promote the release of inflammatory factors by other immune cells, exacerbating inflammatory damage to tissues. Utilizing IL-1ß as the detection index to find small-molecule inhibitors targeting NLRP3 from natural products will benefit the search for drugs for inflammation-related diseases. During the exploration of anti-inflammatory active components derived from the flowers of Dolichos lablab L., an ingredient in traditional Chinese medicine with dual applications in both medicinal treatment and dietary consumption, fourteen compounds (1-14), including seven previously unreported ones, named flosdolilabnitrogenousols A-D (1-4) and flosdolilabsaponins A-C (5-7), were found. Their structures were established through extensive NMR spectra determination, HR-ESI-MS analysis, ECD calculations, and chemical reactions. Flosdolilabsaponin A (5) stands out as an exceptionally rare tetracyclic lactone oleane-type saponin. Additionally, the inhibitory activity on IL-1ß release of all compounds, without cytotoxicity, was evaluated using BMDMs stimulated with LPS/Nigericin. An Elisa assay revealed that compounds 1, 8, 9, and 11-14 exhibited significant inhibition of IL-1ß release at a concentration of 30 µM. Structure-activity relationships were also discussed. This study indicates that D. lablab flowers possess anti-inflammatory activity, which might exert its effect by suppressing the activation of the NLRP3 inflammasome.

Pesticide Contamination in Native North American Crops, Part II-Comparison of Flower, Honey Bee Workers, and Native Bee Residues in Lowbush Blueberry.

In lowbush blueberry fields, we conducted residue analysis comparing flowers, trapped pollen (honey bee and Osmia spp.), and collected bees (honey bee workers, bumble bee queens, and non-Bombus spp. wild native bees). The study was conducted from 2012 to 2014. The number of pesticide residues, total concentrations, and risk to honey bees (Risk Quotient) on flowers were not significantly different from those determined for trapped honey bee pollen (except in one study year when residues detected in flower samples were significantly lower than residue numbers detected in trapped pollen). The compositions of residues were similar on flowers and trapped pollen. The number of residues detected in honey bee pollen was significantly greater than the number detected in Osmia spp. pollen, while the total concentration of residue was not different between the two types of pollen. The risk to honey bees was higher in trapped honey bee pollen than in trapped Osmia spp. pollen. The analysis of honey bee workers, native bumble bee queens, and native solitary bees showed that although more pesticide residues were detected on honey bee workers, there were no differences among the bee taxa in total residue concentrations or risk (as estimated in terms of risk to honey bees).

Mosquito pollination of plants: an overview of their role and an assessment of the possible contribution of disease vectors.

Mosquitoes visit flowers to obtain sugar or other nutrients and therefore possibly serve as major or minor pollinators of some plant species. They also often derive plant nutrients from other sources, such as extrafloral nectaries and honeydew. In a few cases, the plant-mosquito relationship is close, and mosquito pollination has been confirmed. Most plant species visited by mosquitoes, however, appear to depend on multiple means of pollination, particularly other flower-feeding insects. In addition, most mosquito species visit the flowers of many kinds of plants, possibly dispersing pollen in both biologically meaningful and irrelevant ways. This apparent lack of selectivity by both plants and mosquitoes liberates each of them from dependence on an unreliable pollen vehicle or nutrient source. A hypothetical pollinating role for the two top vectors of devastating human-disease pathogens, Anopheles gambiae or Aedes aegypti, relies on indirect evidence. So far, this evidence suggests that their participation in pollen transfer of native, introduced, or beneficial plants is negligible. The few plant species likely to be pollinated by these vectors are mostly invasive, harmful weeds associated with humans. That conclusion draws support from four characteristics of these vectors: (1) the numerous alternative potential pollinators of the flowers they visit; (2) their common use of diverse non-floral sources of nutrients; (3) the females' infrequent sugar feeding and heavy reliance on human blood for energy; and (4) their relatively low population densities. From these traits it follows that focused suppression or elimination of these two vectors, by whatever means, is highly unlikely to have adverse effects on pollination in endemic biotic communities or on ornamental plants or food crops.

Identification of candidate genes associated with double flowers via integrating BSA-seq and RNA-seq in Brassica napus.

As a Brassica crop, Brassica napus typically has single flowers that contain four petals. The double-flower phenotype of rapeseed has been a desirable trait in China because of its potential commercial value in ornamental tourism. However, few double-flowered germplasms have been documented in B. napus, and knowledge of the underlying genes is limited. Here, B. napus D376 was characterized as a double-flowered strain that presented an average of 10.92 ± 1.40 petals and other normal floral organs. F1, F2 and BC1 populations were constructed by crossing D376 with a single-flowered line reciprocally. Genetic analysis revealed that the double-flower trait was a recessive trait controlled by multiple genes. To identify the key genes controlling the double-flower trait, bulk segregant analysis sequencing (BSA-seq) and RNA-seq analyses were conducted on F2 individual bulks with opposite extreme phenotypes. Through BSA-seq, one candidate interval was mapped at the region of chromosome C05: 14.56-16.17 Mb. GO and KEGG enrichment analyses revealed that the DEGs were significantly enriched in carbohydrate metabolic processes, notably starch and sucrose metabolism. Interestingly, five and thirty-six DEGs associated with floral development were significantly up- and down-regulated, respectively, in the double-flowered plants. A combined analysis of BSA-seq and RNA-seq data revealed that five genes were candidates associated with the double flower trait, and BnaC05.ERS2 was the most promising gene. These findings provide novel insights into the breeding of double-flowered varieties and lay a theoretical foundation for unveiling the molecular mechanisms of floral development in B. napus.

Simulation analyses of the evolution of intra-inflorescence flowering patterns assuming selection on anthesis interval among individual flowers.

What conditions select flowering patterns within inflorescences, or variation in the anthesis interval within inflorescences among plants? Under what conditions are gradual blooming and simultaneous blooming, both traits related to floral display size, advantageous? We constructed a simulation model in which the opening times and longevities of individual flowers within inflorescences, the sizes of attractive structures of individual flowers, and the numbers of ovules and pollen grains produced by individual flowers evolve. Individual plants in the population compete for pollinators, and plants are selected by pollinators according to their floral display sizes and amounts of resources allocated to attractive structures. We found that, if the proportion of pollen on a pollinator deposited on a stigma was low, gradual blooming did not evolve even if inbreeding depression was greater than 0.5. This is because the amount of outcross-pollen on pollinators decreased at a low rate during flower visits within a single inflorescence, and the selfing rate was suppressed to a low level even if the floral display size was large. On the other hand, if the proportion of pollen deposition was high, gradual blooming evolved even if inbreeding depression was smaller than 0.5. This may be because gradual blooming can enhance pollen delivery to other plants by reducing the loss of self-pollen by geitonogamy. On the other hand, allocation ratios among floral organs (female and male organs and attractive structures) were independent of the degree of simultaneous and gradual blooming within inflorescences. We concluded that the evolution of gradual blooming is more strongly affected by the proportion of pollen on a pollinator deposited on a stigma than by inbreeding depression.

Effects of different cold plasma treatments on chemical composition, phenolics bioaccessibility and microbiota of edible red mini-roses.

This study evaluated the effect of dielectric barrier discharge (DBD) and glow discharge (glow) cold plasma treatments in color, sugars, organic acids, phenolics (concentration and bioaccessibility), antioxidant activity, volatiles, and microbiota of edible mini-roses. Plasma treatments did not affect the flowers' color, while they increased organic acids and phenolics. Flowers treated with DBD had a higher concentration of most phenolics, including hesperidin (84.04 µg/g) related to antioxidant activity, and a higher mass fraction of most volatiles, including octanal (16.46% after 5 days of storage). Flowers treated with glow had a higher concentration of pelargonidin 3,5-diglucoside (392.73 µg/g), greater bioaccessibility of some phenolics and higher antioxidant activity. Plasma treatments reduced the microbiota diversity in mini-roses. Regardless of the plasma treatment, phylum Proteobacteria, family Erwiniaceae, and genus Rosenbergiella were the dominant groups. Results indicate plasma treatments as promising technologies to improve the quality and increase phenolic and specific volatile compounds in mini-roses.

Training kindergarten children on learning from their mistakes.

This study investigated whether feedback on their errors and speed improves kindergarten children's performance in an executive function (EF) task. Children from Switzerland (N = 213, 49% female, Mage = 6.4 years) were tested in the Hearts and Flowers task pre- and post-training and trained either on a variant of this task with (n = 71) or without feedback (n = 72), or on a control learning task (n = 70). The feedback group performed more efficiently than the no-feedback group during the intervention and partially also in the post-test. Both EF training groups performed more efficiently than the control group in the post-test. These results suggest that kindergarten children detect and monitor their errors and even get better at it given the opportunity to practice. Moreover, they benefit additionally from external feedback. Integrating feedback into computerized cognitive training (and learning apps) could be a potential avenue for interventions in school settings.

A novel anti-inflammatory flavonoid from flowers of Hibiscus mutabilis L.

Hibiscus mutabilis L. is a Traditional Chinese Medicinal plant of significant value. However, there has been limited research focusing specifically on its flowers. In this study, we report the isolation of one novel and nine known flavonoids from the flowers of H. mutabilis L. The structures of these compounds were elucidated using chemical and comprehensive spectral analysis, involving 1D, 2D NMR, and HRESIMS. The novel compound was further evaluated for its anti-inflammatory and cytotoxic activities using in vitro assays on RAW264.7 cells. Compound 1 at the concentration of 6.25 µM significantly inhibited the production of NO and TNF-α induced by LPS in RAW264.7 cells, exhibiting superior efficacy compared to the positive control dexamethasone, thus indicating its potential as an anti-inflammatory drug candidate.

Ectopic enhancer-enhancer interactions as causal forces driving RNA-directed DNA methylation in gene regulatory regions.

Cis-regulatory elements (CREs) are integral to the spatiotemporal and quantitative expression dynamics of target genes, thus directly influencing phenotypic variation and evolution. However, many of these CREs become highly susceptible to transcriptional silencing when in a transgenic state, particularly when organised as tandem repeats. We investigated the mechanism of this phenomenon and found that three of the six selected flower-specific CREs were prone to transcriptional silencing when in a transgenic context. We determined that this silencing was caused by the ectopic expression of non-coding RNAs (ncRNAs), which were processed into 24-nt small interfering RNAs (siRNAs) that drove RNA-directed DNA methylation (RdDM). Detailed analyses revealed that aberrant ncRNA transcription within the AGAMOUS enhancer (AGe) in a transgenic context was significantly enhanced by an adjacent CaMV35S enhancer (35Se). This particular enhancer is known to mis-activate the regulatory activities of various CREs, including the AGe. Furthermore, an insertion of 35Se approximately 3.5 kb upstream of the AGe in its genomic locus also resulted in the ectopic induction of ncRNA/siRNA production and de novo methylation specifically in the AGe, but not other regions, as well as the production of mutant flowers. This confirmed that interactions between the 35Se and AGe can induce RdDM activity in both genomic and transgenic states. These findings highlight a novel epigenetic role for CRE-CRE interactions in plants, shedding light on the underlying forces driving hypermethylation in transgenes, duplicate genes/enhancers, and repetitive transposons, in which interactions between CREs are inevitable.