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bioRxiv2026

Subcellular carbohydrate compartmentation and organic acid signatures reveal natural variation in cold acclimation of Arabidopsis thaliana

Plant cold acclimation emerges from coordinated adjustments in photosynthesis, primary metabolism, and intracellular carbon allocation. Yet, the regulatory role of subcellular metabolite compartmentation in natural variation of cold acclimation remains insufficiently understood. Here, we investigated four Arabidopsis thaliana accessions grown either individually or in bulk to determine how growth configuration and genotype shape the metabolism of sugars and organic acids during cold exposure. Using non-aqueous fractionation, we quantified plastidial, cytosolic, and vacuolar sugar pools alongside whole-cell carbohydrates, organic acids, enzyme activities, photosynthetic parameters, and stress markers. A neural-network classifier revealed that subcellular sugar distribution together with sugar amounts and organic acids provided the strongest discriminatory power among accessions, surpassing photosynthetic traits and enzyme activities. Our findings demonstrate that natural variation in cold acclimation is strongly determined by genotype-specific subcellular metabolite architectures, and that the cultivation strategy modulates these intracellular signatures. We conclude that subcellular compartmentation of metabolites represents a cellular control layer for natural variation of cold acclimation and resilience in Arabidopsis thaliana.

plant biology↗

bioRxiv2026

Extracellular Vacuole-derived bodies (EVacs) mediate RNA secretion in plants

Extracellular RNAs are found in the plant extracellular space, but how they are exported from cells remains unclear. We found that the plant vacuole is a major source of extracellular RNA and identified a class of large extracellular vacuole-derived bodies, which we termed EVacs, that are key mediators of this transport. EVacs are marked by the vacuolar membrane (tonoplast) proteins {gamma}-TIP and V-ATPase and originate as intravacuolar structures formed by inward folding of the tonoplast, encapsulating intact cytoplasmic material, including both RNAs and proteins. These intravacuolar bodies then escape the vacuole and are subsequently released from the plasma membrane of mesophyll cells into the apoplast. These findings provide a novel mechanism for the unconventional secretion of macromolecules in plants.

plant biology↗

bioRxiv2026

Rate of meristem initiation driven by the MADS-WUS axis contributes to floral survival and inflorescence evolution in grasses

Crop domestication has repeatedly shaped inflorescence architecture to improve floral production, but mechanisms coordinating the rate of floral initiation, maturation and survival remain unclear. Combining morphometry, modelling and molecular genetic analyses, we show that floral production in the indeterminate barley (Hordeum vulgare L.) inflorescence follows an "initiate fast-die young" strategy orchestrated by a main MADS-box gene, SPIKELET INITIATION AND FERTILITY (SIF). SIF accomplishes this duality by coordinately terminating the inflorescence meristem via WUSCHEL and activating the floral meristem via APETALA1 (Vrn-H1). Hereby, the ancestral SIF "slow" allele promotes a timely commitment to floral maturation, whereas the derived "fast" allele permits more floral initiations. Postdomestication selection of SIF alleles thus enables diversified reproductive strategies in barley populations to maintain yield traits in the field. Finally, we show that a lineage-specific SIF duplication contributed to meristem fate transition and inflorescence evolution during Triticeae cold adaptation. Our results establish developmental rate as a key driver of architectural innovation and reproductive success.

plant biology↗

bioRxiv2026

Breeding cassava for intercropping with cowpea: monoculture selection captures most intercrop selection gain, but targeted testing remains necessary

Intercropping dominates smallholder cassava production in sub-Saharan Africa, yet cassava breeding programs evaluate genotypes exclusively under monoculture. Despite consistently reported system-level yield advantages, cassava yield is reduced by 17 to 51% under intercropping, indicating a need to reduce this competitive disadvantage through breeding. However, the quantitative-genetic foundations of intercrop breeding remain uncharacterized for tropical root crop systems. We hypothesized that monoculture selection would capture most, but not all, genetic merit for intercropping and that a limited tester set would be sufficient if general mixing ability predominated. We evaluated 120 cassava clones previously selected under monoculture in IITA advanced yield trials, testing them under monoculture and in intercrop with two contrasting cowpea varieties across two years at Ibadan, Nigeria. Spatial mixed models quantified genetic variation, genotype by cropping system interaction, cross system genetic relationships, realized selection gain, mixing ability, tester effects, and land equivalent ratio. Intercropping reduced cassava fresh root yield by 19%, but total land equivalent ratios exceeded 1.0 for all clones, confirming a system-level land use advantage. Cassava performance under intercropping was heritable, with estimates of 0.50 to 0.75, and genotype by cropping system interaction was not significant. Genetic correlations between monoculture and intercrop performance were high and approached unity (rg = 0.92 to 0.99), and selection efficiency was 26 to 44% at 10% intensity, confirming that high genetic correlation does not guarantee effective indirect selection. Monoculture selection captured approximately two-thirds of direct intercrop gain. General mixing ability dominated, specific mixing ability was negligible, and producer effects explained 20 to 47% of intercrop variance. The two architecturally and phenologically contrasting cowpea varieties had limited influence on cassava rankings. Here, we show for the first time that cassava breeding for intercropping can retain monoculture selection during early stages while adding two representative cowpea testers at the advanced trial stage. This staged strategy aligns cassava breeding with diversified smallholder systems without creating a separate pipeline.

plant biology↗

bioRxiv2026

Nitrate regulates anchor root development

Nitrogen is a critical nutrient necessary for plant growth and survival. Plasticity in root architecture helps adapt to soil nitrogen levels for optimal nitrogen uptake; the nitrate form of soil nitrogen is a major modulator of root architecture. Although details of nitrate-regulated primary and lateral root growth are known, nitrate-regulated formation of anchor roots, which arise from the collet, is not understood. In this work, we uncover a role for nitrate in the regulation of anchor root formation. We find that cytokinin inhibits anchor root formation with rising nitrate. These cytokinin effects on anchor root formation rely on regulated indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA) conversion. These data point toward a mechanism by which nitrate controls a previously underappreciated aspect of nitrate-dependent root architecture driven by anchor roots.

plant biology↗

bioRxiv2026

DIFFERENTIAL PHOTOSYNTHETIC RESPONSES TO GLUFOSINATE AMMONIUM IN TWO GRASS WEEDS: Lolium multiflorum AND Echinochloa crus-galli.

Background: Weed control is one of the main challenges in agriculture today, particularly due to the increasing occurrence of herbicide-resistant populations. Among the most problematic species are Lolium multiflorum (L.) and Echinochloa crus-galli (L.) Beauv., for which glyphosate-resistant populations have been reported. In this context, glufosinate ammonium has emerged as an alternative for their control; however, its efficacy may vary depending on species and photosynthetic metabolism. Objective: The objective of this study was to evaluate the differential sensitivity of ryegrass (C3) and barnyardgrass (C4) to ammonium glufosinate by analyzing physiological responses associated with leaf senescence and photosystem II activity. Methods: Visual injury, chlorophyll fluorescence, and ammonium accumulation were assessed. Results: Results revealed a differential response between species. Barnyardgrass exhibited earlier symptom onset and a greater reduction in the quantum yield of photosystem II ({Phi}PSII), whereas ryegrass showed a slower senescence process. These differences indicate a higher sensitivity of barnyardgrass to glufosinate ammonium, possibly associated with its C4 photosynthetic metabolism. Conclusions: It is concluded that the effectiveness of glufosinate ammonium depends on the type of photosynthetic metabolism and on the ability of each species to cope with herbicide-induced oxidative stress. This information contributes to optimizing glufosinate ammonium use and to the development of management strategies aimed at delaying the evolution of herbicide resistance.

plant biology↗

bioRxiv2026

Arabidopsis Acyl-CoA Binding Protein 4, ACBP4, functions in developmentally programmed endoreduplication

Powdery mildew fungi induce localized endoreduplication, a variant of the cell cycle in which DNA is replicated but cells do not divide, in leaf mesophyll cells underlying the fungal feeding structure. Induced endoreduplication occurs concurrent with powdery mildew (PM) spore production and is associated with enhanced metabolic capacity and flux to lipids. The final ploidy of these cells is highly correlated with fungal spores produced and is the consequence of both basal (developmental) ploidy and PM-induced endoreduplication programs. Herein, we find the Arabidopsis lipid trafficking and regulatory protein ACYL-COA BINDING PROTEIN 4 (ACBP4) enhances PM spore production on Arabidopsis leaves. ACBP4 does not limit plant defense but instead supports basal mesophyll cell ploidy, with decreased final ploidy in cells underlying the fungal feeding structure in acbp4 mutants compared to wild-type (WT). Leaf epidermal cell size is decreased and stomatal density is increased in acbp4, consistent with a role for ACBP4 in developmentally programmed endoreduplication. Moreover, hypocotyl elongation in the dark, which is driven by programmed developmental endoreduplication, shows reduced hypocotyl length, cell length and ploidy in acbp4 versus WT. Together, our findings establish a novel means by which a plant ACBP promotes cell metabolism and development, with potential applications to agricultural productivity and quality.

plant biology↗

bioRxiv2026

A single dsRNA spray silences VAMT and shifts habanero pepper fruit metabolism towards capsinoids

Capsaicinoids are synthesized in the placenta of Capsicum fruit, where vanillylamine aminotransferase (VAMT) catalyzes the formation of vanillylamine, the precursor of the pathway. The modulation of pungency has relied on genetic breeding and transgenic approaches, and this pathway has not been addressed by spray-induced gene silencing. The aim of this study was to evaluate whether a single non-invasive spray of double-stranded RNA (dsRNA) targeting VAMT allows the gene to be silenced and capsaicinoid accumulation to be modified in Capsicum chinense fruit. The molecule was designed in silico and applied at 10 days post-anthesis. Pedicel injection reduced the VAMT transcript in a dose-dependent manner, with three levels of inhibition distinguishable from one another. Spraying with surfactant reduced it by 86.2 %, a magnitude statistically indistinguishable from the 90.6 % obtained by injection, and also reduced the Pun1 transcript, a co-regulation previously described only as a difference between cultivars. Analysis by gas chromatography coupled to mass spectrometry showed reductions of 84.4 % in capsaicin and 68.5 % in dihydrocapsaicin, the loss of nonivamide and one further vanillylamine-derived compound, and the detection of capsiate and a second capsinoid, absent in control fruits. The siRNA was detected in non-treated tissues, and a single topical application is therefore sufficient to silence an endogenous biosynthetic gene and shift the metabolic profile of the fruit without genetic modification.

plant biology↗

bioRxiv2026

Implementation and calibration of the Vaganov-Shashkin model in the virtualRings R package

Process-based tree growth models provide a mechanistic framework for investigating how climate conditions regulate tree growth across daily to annual time scales. Yet, their broader application across species and environments is constrained by the limited accessibility in open-source environments and the difficulty of estimating physiological parameters that are rarely measured directly. Here, we present virtualRings, a new R package integrating the Vaganov-Shashkin model (VSM) and the RINGS3 models, and focus on the implementation and calibration of VSM. Using tree-ring width observations from seven Northern Hemisphere sites across various environmental conditions, we compared the traditional bootstrap-based calibration approach with the Covariance Matrix Adaptation Evolution Strategy (CMA-ES). CMA-ES improved agreement between simulated and observed radial tree growth and provided an efficient approach for model parameter estimation. We further evaluated practical CMA-ES settings to balance computational cost and performance and discussed its potential limitations. The virtualRings package provides an open and reproducible platform for tree growth simulation, facilitating the application of important process-based models across species and environments and the investigation of how temperature and moisture constraints regulate daily tree-ring formation across spatial and temporal scales.

plant biology↗

bioRxiv2026

Timing of transient darkness shapes carbon-nitrogen metabolism and sugar signaling in sugarcane

Fluctuating light is common in field environments. Yet, the mechanisms by which C4 crops coordinate carbon and nitrogen metabolism during short-term carbon deprivation remain poorly understood. Here, we imposed transient darkness at different phases of the diel cycle to assess how the timing of light loss affects photosynthesis, carbohydrate turnover, amino acid dynamics, and sugar-sensing pathways in commercial sugarcane leaves. Early-day darkness significantly impaired photosynthetic induction and revealed a temporal disconnect between stomatal and metabolic limitations, whereas midday and late-day treatments caused temporary, time-specific disruptions in carbon assimilation. These shifts altered the balance between sucrose preservation and catabolic mobilization, leading to treatment-dependent changes in starch reserves and free amino acids. Core circadian components largely maintained their phase relationships, but their amplitudes varied across treatments, consistent with partial decoupling from carbon status. Darkness also reorganized energy signaling, with SnRK1 and DIN6 responses associated with greater declines in sucrose. Notably, trehalose-pathway transcripts showed marked changes in network connectivity, with ScTPSIIG consistently emerging as a highly connected candidate associated with photosynthetic performance, water-use traits, sugar sensing, and amino acid metabolism. Overall, these results indicate that the timing of carbon limitation and residual sucrose availability shape distinct metabolic responses, while trehalose metabolism provides a candidate regulatory layer coordinating carbon-nitrogen adjustment during the diel cycle, highlighting class II TPS proteins as targets for functional investigation of metabolic resilience in sugarcane.

plant biology↗

bioRxiv2026

A CO2-limitation-induced cytosolic repressor enables shutdown of the algal CO2-concentrating mechanism

Aquatic photosynthetic organisms face limited CO2 availability because CO2 diffuses slowly in water and most dissolved inorganic carbon (Ci) exists as HCO3- at physiological pH. To overcome this limitation, aquatic photoautotrophs operate CO2-concentrating mechanisms (CCMs) that elevate CO2 around Rubisco and sustain carbon fixation. Because CCM operation consumes energy, it must be suppressed when CO2 becomes abundant, but how this shutdown occurs remains poorly understood. In Chlamydomonas reinhardtii, the nuclear protein CBP1 was identified as a CCM repressor, but its loss causes only partial derepression under high CO2, indicating that an additional mechanism is required for complete shutdown. Here, we identify High-Affinity CCM Repressor 1 (HCR1), a cytosolic protein related to CBP1, as a second repressor. Under high CO2, hcr1 mutants retained high affinity for Ci and derepressed CCM and photoacclimation genes. Combined disruption of HCR1 and CBP1 further increased Ci affinity, approaching that of wild-type cells with a fully induced CCM under CO2 limitation, and promoted the accumulation of Ci transporters. HCR1 loss also prevented redistribution of the chloroplast regulator CAS away from the pyrenoid and was accompanied by retention of a pyrenoid starch sheath. In contrast, LCIB, a chloroplast CO2-recapture protein, relocated normally. Unexpectedly, HCR1 accumulated during CO2 limitation and declined after transfer to high CO2. These results show that CCM shutdown is an active transition rather than the passive reversal of induction. We propose that CBP1 restrains CCM1-dependent transcription, while HCR1 is preloaded during CO2 limitation to terminate the CAS-associated, starch-sheathed, high-affinity state when CO2 becomes replete.

plant biology↗

bioRxiv2026

Bioengineering of Pea (Pisum sativum) for the Expression of Myoglobin, a Heme-containing Animal Protein

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

plant biology↗

bioRxiv2026

Impact of Water Deficit on Growth, Biochemical, and Physiological Traits in Eggplant MAGIC Lines

Climate change exacerbates agricultural water scarcity, necessitating the development of drought-tolerant crop varieties. This study evaluates 12 eggplant lines from a MAGIC (Multi-parent Advanced Generation Intercross) population, previously selected for contrasting responses to water deficit during the vegetative stage. To validate tolerance under adult production conditions, plants underwent five irrigation-withholding cycles over a 170-day greenhouse growing period. Yield components, the Stress Tolerance Index (STI), and physiological parameters (water status and stomatal conductance) were evaluated. Additionally, photosynthetic pigments, oxidative stress markers, antioxidant compounds, and osmolytes were quantified to characterize the biochemical basis of tolerance alongside final biomass production. The results showed that four of the five lines that were previously classified as tolerant in the vegetative stage remained among the most tolerant at the reproductive stage. Specifically, lines L13, L78 and L179 were the most productive under water-limited conditions. While L13 and L179 exhibited stable tolerance throughout all developmental stages, L78 displayed stage-specific tolerance, manifested only during the reproductive growth phase. These findings emphasise the importance of integrating early-stage screening with adult-stage validation in order to capture the full spectrum of genetic drought tolerance. The most productive lines were characterised by moderate aboveground biomass, high leaf hydration and maintained stomatal conductance. However, the strategies employed differed: while L179 exhibited high photosynthetic pigment content, L13 was characterised by high total sugar accumulation. Overall, these results provide a multi-trait roadmap and identify elite MAGIC parental lines for breeding climate-resilient eggplant cultivars.

plant biology↗

bioRxiv2026

Loss of ELM1B impairs mitochondrial fission, matrix redox state and stress tolerance in Physcomitrium patens

Mitochondria are endosymbiont-derived organelles that play a central role in cellular metabolism, energy production and stress responses. While single mitochondria represent functional units, they continuously exchange their contents through fusion and fission, facing stress conditions as a dynamic population. To date, it remains largely unknown how stress alters mitochondrial dynamics in plants and how altered dynamics affect mitochondrial properties and plant stress resilience. Here, we investigate mitochondrial dynamics in response to oxidative stress in the non vascular model plant Physcomitrium patens. By creating mutants with impaired mitochondrial fission in different reporter lines for mitochondrial parameters, we additionally analyse effects of chronic changes to mitochondrial population dynamics. We found that Mito-Paraquat (MtPQ) treatment increased the glutathione redox potential EGSH in mitochondria, the cytosol and chloroplasts, as monitored via roGFP2-based genetically encoded biosensors. Mitochondria elongated within hours and showed a concomitant and heterogenous increase of matrix EOSred, that we propose as a marker for matrix protein damage. Mitochondrial fission mutants lacking PpELM1B (ELONGATED MITOCHONDRIA) displayed distinct changes of mitochondrial morphology parameters as determined by automated 3D-segmentation and feature mapping (MorphoMapper) of confocal z-stacks. Elongated mitochondria in Ppelm1bge lines showed an oxidative matrix EGSH shift and increased matrix EOSred while matrix mixing still occurred, albeit at the same slow rate as in wildtype, within days. Macroscopically, Ppelm1bge lines displayed reduced growth, decreased respiration, and a higher sensitivity to oxidative stress. Our results show that plant mitochondrial morphology and physiological parameters specifically shift in response to stress and impaired fission. Mitochondrial fission is vital to maintain a healthy mitochondrial population that sustains plant oxidative stress tolerance.

plant biology↗

bioRxiv2026

Salicylic acid-triggered apoplastic proteolysis releases cryptic phytocytokines with distinct immunogenic functions

Plants rely on an innate immune system to defend against pathogens through various molecular responses. In addition to classical damage- and pathogen-associated molecular patterns (DAMPs and PAMPs), plants produce endogenous signaling peptides termed phytocytokines that amplify and regulate immune responses following stress. Although most characterized phytocytokines originate from dedicated precursor proteins, the contribution of multifunctional proteins to phytocytokine generation remains poorly understood. Here, we show that salicylic acid (SA) rapidly remodels the maize apoplastic peptidome through an early, transient proteolytic program driven by apoplastic serine hydrolases. Time course peptidomics identified fourteen candidate phytocytokines, including two cryptic peptides, PC13 and PC14, released from the stress-associated zinc-finger protein ZmSAP7 and the migration inhibitory factor-like protein ZmMDL1, respectively. Both peptides activated immune-associated gene expression but triggered distinct transcriptional responses and exerted opposing effects on Ustilago maydis infection, with PC13 enhancing resistance and PC14 promoting susceptibility. Biochemical analysis demonstrated that PMSF-sensitive apoplastic serine proteases directly process ZmMDL1 to release PC14. Together, our findings uncover a SA-responsive proteolytic pathway that generates functionally distinct phytocytokines from multifunctional proteins, expanding the repertoire of immune signaling peptides and revealing an additional layer of regulation in plant defense.

plant biology↗

bioRxiv2026

Effects of spectral light quality on growth, photosynthetic pigments and bioactive compounds in Brassicaceae microgreens

LED spectral composition is an important tool for improving the growth and nutritional quality of microgreens cultivated in controlled environments. This study evaluated the effects of three LED light treatments on growth, morphology, pigments, primary metabolites, phenolic composition, and antioxidant capacity in arugula (Eruca sativa), mustard (Brassica juncea), and radish (Raphanus sativus) microgreens. Microgreens were cultivated under controlled environmental conditions and exposed to broad-spectrum white (W), blue-enriched white (WB), and red-enriched white (R) light at a photosynthetic photon flux density of 200 micromol/m2/s. Light quality did not affect yield in any species. However, R increased cotyledon area in arugula by 50 to 60% and promoted hypocotyl elongation in both arugula and radish, whereas W resulted in the longest hypocotyls in mustard. Photosynthetic pigment composition responded differently among species. In mustard, WB increased the chlorophyll a/b ratio (1.12 to 1.18), whereas lutein concentration decreased from 7.06 to 4.20 mg 100 g/FW. Primary metabolism also responded to light treatments in a species-dependent manner. In mustard, W increased glucose (0.43 vs. 0.26 and 0.29 g 100 g/ FW) and fructose (0.33 vs. 0.20 and 0.22 g 100 g/ FW) concentrations compared with WB and R. Organic acid composition was more responsive to light treatments in radish, with higher concentrations under R. Phenolic metabolism also responded in a species-dependent manner. In mustard, W increased total phenolic content to 0.25 mg GAE g/FW compared with 0.15 mg GAE g/FW under WB and R, and ABTS antioxidant capacity to 1.17 mg TE g/FW compared with 0.74 and 0.75 mg TE g/FW under WB and R, respectively. Individual phenolic compounds were also affected by light treatments, particularly in arugula and mustard. These findings demonstrate that the effects of LED spectral composition on microgreen quality are highly species-dependent. Therefore, LED light spectra should be optimized according to the target species and the desired quality attributes rather than applying a single lighting strategy to all Brassicaceae microgreens.

plant biology↗

bioRxiv2026

Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTORs are novel susceptibility factors for Colletotrichum higginsianum

Colletotrichum higginsianum (Ch) is a hemibiotrophic fungal pathogen that infects Brassicaceae plants, including Arabidopsis thaliana. The molecular mechanisms underlying the Ch-A. thaliana interaction are not fully understood. Particularly, the susceptibility factor against Ch infection remains to be determined. Here, we report that A. thaliana ACTIN DEPOLYMERIZING FACTORs (ADFs), ancient proteins that regulate the organization and dynamics of actin filaments (AFs), function as susceptibility factors during Ch infection. Among 11 ADFs encoded in A. thaliana genome, subclass I ADFs that include ADF1, -2, -3, and -4, express throughout the plant. We found that knockout mutant of ADF4 and transgenic plants in which the expression of all of subclass I members is suppressed (ADF1-4Ri) exhibited increased resistance to Ch. Cytological analyses revealed that both Ch penetration and secondary hyphae formation were suppressed in adf4 and ADF1-4Ri. This enhanced resistance was associated with suppression of Ch-induced AF fragmentation. In addition, we found that PENETRATION 2 (PEN2) plays a critical role in the Ch resistance in adf4 and ADF1-4Ri. Our findings suggest that subclass I ADFs promote AF fragmentation during Ch infection, thereby suppressing PEN2-associated mitochondria accumulation at Ch entry sites. Together, these results raise the possibility that Ch exploits host ADF-dependent actin regulation to facilitate successful infection.

plant biology↗

bioRxiv2026

VLCFA-mediated inter-cell layer communication controls cellular pluripotency in Arabidopsis callus

Plants have remarkable capacity to reconstruct entire organ systems from tissue explants. In Arabidopsis two-step tissue culture system, pluripotency regulators are specifically expressed in the middle-cell layer of the stratified callus tissue. However, regulatory mechanisms underlying the radial patterning of callus remained unclear. Here, we found that very-long-chain fatty acids (VLCFAs) synthesized in the epidermis-like outermost layer are essential for pluripotency acquisition and successful shoot regeneration. Our genetic and transcriptomic analyses revealed that the regulatory roles of VLCFAs on pluripotency acquisition involve inter-cell layer signaling in callus tissue, while they are at least partly independent of ATML1/PDF2 functions and cuticular wax synthesis in the outermost layer. VLCFAs spatially restrict procambium cell identity by non-cell-autonomously suppressing cytokinin signaling, thereby allowing for establishment of the middle-cell layer. We propose that the inhibitory relationships between layer-specific regulators underlie the intricate balance of cellular fate determination in pluripotent callus.

plant biology↗