Biology subjects

Fladung, M.

Publications and source records attributed to Fladung, M..

8 recordsLinked to original sources

bioRxiv2026

A heat-assisted ttLbCas12a workflow incorporating crRNA 3'-tailing enables efficient multiplex biallelic genome editing in hybrid poplar

In forest trees, high genome-editing efficiency in primary transformants is particularly important for functional genomics and trait improvement, as long generation times severely limit the use of subsequent generations to recover fully edited genotypes. This challenge is further compounded in hybrid poplar, where paralogous gene copies and allelic variation make efficient multiplex editing particularly difficult. We optimized LbCas12a-mediated editing in Populus x canescens (Populus tremula x Populus alba), clone INRA 717-1B4, by targeting four paralogous PtaABA8ox4 loci using temperature-tolerant ttLbCas12a, a two-stage heat treatment, and crRNA 3'-tailing. The heat-treatment regime preserved transgenic recovery, while ttLbCas12a-based constructs and heat treatment substantially increased biallelic editing. All seven lines in the highest-performing group, combining ttLbCas12a, 3'-tailed crRNAs, and heat treatment, were biallelically edited at all four targets. crRNA 3'-tailing altered predicted secondary structures and showed a consistent numerical tendency toward improved editing, but its independent effect was not statistically resolved. Mutations were dominated by small deletions, and biallelic out-of-frame recovery increased in ttLbCas12a-based contexts without scaling proportionally with biallelic editing. Thus, heat-assisted ttLbCas12a-mediated approaches enable efficient multiplex editing in hybrid poplar, whereas genotype quality remains dependent on repair outcome and somatic uniformity.

plant biology↗

bioRxiv2026

CRISPR/Cas9 knockout of PtaAGL16 improves post-drought recovery and shoot vitality in hybrid poplar

Drought stress severely limits tree growth and survival, a problem intensifying under climate change due to the rising frequency of extreme weather events. Identifying genetic variants that modulate drought stress responses and enhance resilience is therefore of great interest for developing climate-adapted trees. While the MADS-box transcription factor AGL16 has been implicated in drought responses and stomatal regulation in herbaceous species, the function of its homolog in woody perennials remains largely unexplored. Here, we investigate the role of PtaAGL16 in the poplar hybrid Populus tremula x P. alba. We generated CRISPR/Cas9 knockout lines of PtaAGL16 in the poplar clone INRA 717-1B4. Plants were subjected to a severe drought stress regime followed by a re-watering recovery phase. Morphological and physiological traits, including growth, stomatal conductance, shoot vitality, and stomatal density, together with stress-associated metabolites and drought-responsive gene expression, were analyzed. All agl16 knockout lines exhibited significantly reduced drought-induced damage compared to the wild-type. While the wild-type suffered extensive defoliation and persistent stem necrosis, the knockout lines retained higher shoot vitality and resumed growth after re-watering, including the formation of new leaves. Consequently, vitality in the knockout lines reached up to 86%, compared to only 34% in the wild-type. This improved resilience was accompanied by reduced persistent oxidative membrane damage, as indicated by lower MDA levels after recovery. These findings indicate that the disruption of PtaAGL16 is associated with enhanced drought resilience in a woody perennial species and highlight its potential as a genetic target for improving climate adaptation in fast-growing forest trees.

plant biology↗

bioRxiv2026

First report of genetic transformation and CRISPR/Cas12a-mediated gene editing of European beech (Fagus sylvatica L.)

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 a 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% within distinct seasonal windows. Protoplast yield and transformation efficiency showed pronounced temporal variation throughout the year, indicating a strong seasonal influence on reproducibility of the workflow despite controlled growth conditions. A basic molecular toolkit for functional genomics and future biotechnological applications was 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). Although further optimization of transformation reproducibility and regeneration systems remains necessary, the presented protoplast platform provides a valuable foundation for transient functional assays and genome editing studies in this non-model tree species.

plant biology↗

bioRxiv2026

Overexpression of PtaHDG11 enhances drought tolerance and suppresses trichome formation in Populus tremula x Populus alba

Extended drought periods are increasingly impacting the growth and survival of forest trees in Central Europe, underscoring the need for innovative approaches to adapt trees to drought. Understanding the functions of genes involved in drought tolerance can contribute to the development of effective strategies to improve drought resilience. Although the homeobox-leucine zipper protein HDG11 has been identified as a promising target for enhancing drought stress tolerance in plants, the functional role of the native HDG11 gene in tree species remained uncharacterized. To address this gap, we identified the homolog of the AtHDG11 gene in the poplar hybrid Populus tremula x Populus alba, clone INRA 717-1B4, and subsequently transformed the PtaHDG11 gene into this clone for constitutive overexpression. Drought stress experiments revealed significantly enhanced drought stress tolerance in PtaHDG11-overexpressing poplars compared with the wild type. This improved tolerance was characterized by higher leaf relative water content, reduced leaf shedding, lower malondialdehyde accumulation and higher expression of antioxidant-related genes, including SOD and CAT. Improved tolerance resulted in significantly increased dry biomass after the recovery phase. Notably, the transgenic poplars exhibited altered basal expression of the cell wall-related genes PtaXTH32-like and PtaEXPA15-like under control conditions, indicating a modified cell wall-associated regulatory state before stress exposure. In addition, PtaHDG11 overexpression was associated with a glabrous phenotype lacking trichomes, pointing to functions of PtaHDG11 beyond drought stress responses in a woody perennial. Together, these findings suggest that PtaHDG11 can contribute to improving drought resilience in trees, with implications for sustainable forest management and breeding programs.

plant biology↗

bioRxiv2026

Male-biased triploidy in Populus tremuloides and genomic structure of its southern relict populations

PremiseIsolated populations at the southern range edge of aspen (Populus tremuloides Michx.) offer a unique opportunity to study ploidy, clonality and distribution of sex under arid, high-elevation conditions. We aimed to characterize such traits, including potential sex biases and associations with ploidy, investigating previously unexamined Texan populations in a range-wide genomic framework of genetic structure and demographic history of this keystone species. MethodsWe combined new genotypic data from western Texas (Davis Mountains, Big Bend, Guadalupe Mountains) with preexisting datasets, assessing range-wide genetic diversity, clonality, ploidy, and sex. We further conducted ADMIXTURE and phylogenetic analyses, reconstructed historical effective population sizes (Ne) with Stairway Plot 2. ResultsTexan stands revealed high clonality, with diploid and triploid genets. Most Davis and Guadalupe Mountain individuals clustered with a southwestern U.S. lineage of aspen, whereas Big Bend individuals grouped with a Mexican, demonstrating for the first time the continuous northward geographic distributions of allele frequencies in aspen coherent with phylogenetics. The significant male bias in sex ratios, particularly among triploids, suggests dimorphism in survival or reproduction. No effect of elevation on sex was identified. Demographic inference indicated an ancient bottleneck ([~]1-2 Mya) common to all six lineages, but more recent historical Ne trajectories differ between the northern and southern regions. ConclusionsThese findings shed light on how clonality, ploidy, and sex have interacted in shaping dioecious plant species, highlighting the importance of incorporating such information into projections of how climate change and habitat loss will affect their distribution, abundance, and the extinction risk of their marginal populations.

evolutionary biology↗

bioRxiv2025

LysM Receptor Proteins are Required for Ectomycorrhizal Symbiosis in Poplar

Successful symbiotic associations often rely on the perception of specific microbial signals by host plants through pattern recognition receptors. LysM receptor-like kinases are key receptors in both rhizobial and arbuscular mycorrhizal symbioses. In this study, we provide the first evidence that such LysM receptor-like kinases also control the establishment of ectomycorrhizal associations in poplar. Using CRISPR/Cas9-mediated genome editing, we knocked out three NFP-like candidate genes in various combinations. We examined their roles in the perception of the ectomycorrhizal fungus Laccaria bicolor, as well as chitin oligomers and lipo-chitooligosaccharides. We demonstrate that all three receptors are necessary for the recognition of symbiosis-related signals, calcium spiking, symbiotic gene expression, and ectomycorrhizal root phenotypes.

plant biology↗

bioRxiv2023

Genomic variation of a keystone forest tree species reveals patterns of local adaptation and future maladaptation

Local adaptation is key for ecotypic differentiation and species evolution. Understanding the underlying genomic patterns can allow the prediction of future maladaptation and ecosystem stability. Here, we report the whole-genome resequencing of 874 individuals from 100 range-wide populations of European beech (Fagus sylvatica L.), one of the most important forest tree species in Europe. We show that genetic variation closely mirrors geography with a clear pattern of isolation-by-distance. Genome-wide analyses for genotype-environment associations (GEAs) identified relatively few potentially adaptive variants after correcting for an overwhelming signal of statistically significant but non-causal GEAs. We characterized the single high confidence genomic region and pinpoint a candidate gene possibly involved in winter temperature adaptation acting by modulating spring phenology. Surprisingly, however, allelic variation at this locus did not result in any apparent fitness differences in a common garden. More generally, reciprocal transplant experiments across large climate distances demonstrated extensive phenotypic plasticity. Nevertheless, we find indications of polygenic adaptation which may be essential in natural ecosystems. This polygenic signal exhibits broad- and fine-scale variation across the landscape highlighting the relevance of spatial resolution. In summary, our results emphasize the importance but also exemplify the complexity of employing natural genetic variation for forest conservation under climate change.

plant biology↗

bioRxiv2021

Nonmuscle myosin IIA dynamically guides regulatory light chain phosphorylation and assembly of nonmuscle myosin IIB

Nonmuscle myosin II minifilaments have emerged as central elements for force generation and mechanosensing by mammalian cells. Each minifilament can have a different composition and activity due to the existence of the three nonmuscle myosin II isoforms A, B and C and their respective phosphorylation pattern. We have used CRISPR/Cas9-based knockout cells, quantitative image analysis and mathematical modelling to dissect the dynamic processes that control the formation and activity of heterotypic minifilaments and found a strong asymmetry between isoforms A and B. Loss of NM IIA completely abrogates regulatory light chain phosphorylation and reduces the level of assembled NM IIB. Activated NM IIB preferentially co-assembles into pre-formed NM IIA minifilaments and stabilizes the filament in a force-dependent mechanism. NM IIC is only weakly coupled to these processes. We conclude that NM IIA and B play clearly defined complementary roles during assembly of functional minifilaments. NM IIA is responsible for the formation of nascent pioneer minifilaments. NM IIB incorporates into these and acts as a clutch that limits the force output to prevent excessive NM IIA activity. Together these two isoforms form a balanced system for regulated force generation.

cell biology↗