Plant vascular systems exhibit a wide developmental spectrum, from rigid woody tissues to soft, fleshy storage tissues. We show that increasing polyamine thermospermine transport into wild‑type rootstocks, together with cytokinin, reprograms xylem identity from woody to fleshy in Arabidopsis. Our findings establish thermospermine as a mobile developmental signal and suggest a strategy for engineering plant vascular architecture. ### Competing Interest Statement Y.H., D.K., and R.R. are inventors on a pending patent application (P37067GB1 BF 30.4.20) covering the results described in this paper. the Academy of Finland, 346139, 336727 the Gatsby Foundation, GAT3395/PR3 the National Science Foundation Biotechnology and Biological Sciences Research Council grant, BB/N013158/1 University of Helsinki, award 7999920 91 EMBO, ALTF 305-2017 ERC, 101166880 Knut and Alice Wallenberg Foundation, KAW 2016.0352, KAW 2020.0240 Swedish Research Council, https://ror.org/03zttf063, VR 2021-04938 ERDF Programme Johannes Amos Comenius, CZ.02.01.01/00/22_008/0004581 National Science Centre Poland, 2022/47/B/NZ9/00558

Abstract Cytokinin (CK) N-glucosides are the most abundant CK metabolites in Arabidopsis and most angiosperms, yet their role in cytokinin activity and response is unclear. Here, we examined metabolomic, transcriptomic, and proteomic profiles of seven CK N-glucoside conjugates in detached Arabidopsis leaves across a 144-hour dark-induced senescence (DIS) timecourse. All tested N-glucosides were found to undergo a slow conversion to their corresponding base forms at position-dependent rates, with N9-glucosides releasing base faster than their corresponding N7-glucosides. Conversion during DIS was strictly isoform-specific and not accompanied by coordinated induction of CK biosynthesis genes, arguing against de novo synthesis as the source of accumulated base. Despite progressive base accumulation, N-glucoside-treated leaves produced substantially fewer Differentially Expressed Genes than direct base application at comparable base concentrations, revealing a disconnect between hormone presence and transcriptional output. Unbiased model comparison identified the base:glucoside ratio as a stronger predictor of CK-Two Component Signaling (TCS) gene expression than absolute base concentration, though modulated by base-type-specific receptor affinities. Early proteomic profiling further revealed a coordinated response shared across N-glucosides but largely absent from base treatments. Together, these findings support that CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms. Highlights Physiology, metabolomic, transcriptomic, and proteomic findings here support CK N-glucosides as kinetically slow, position-dependent reservoirs whose presence in abundance modulate activation of CK-TCS elicited by bioactive forms.

This review highlights recent advances revealing auxin homeostasis as an integrated network of metabolism and transport, emphasizing subcellular compartmentalization as a key regulatory principle. Indole-3-acetic acid (IAA), the principal auxin, is a central regulator of plant growth and development throughout the plant life cycle. Recent discoveries have reshaped our understanding of auxin metabolism and highlighted its close integration with auxin transport in maintaining cellular homeostasis. This review summarizes current knowledge on IAA metabolism and places it into the broader framework of auxin distribution within plant cells. The spatial distribution of auxin is tightly linked to its metabolic regulation, as localized auxin accumulation underlies many developmental and signaling processes. Subcellular compartmentalization of auxin into organelles such as the endoplasmic reticulum or vacuole is closely associated with metabolic modifications that influence auxin transport and availability, although the mechanisms governing the movement of auxin conjugates across organellar membranes remain poorly understood. Directed auxin transport delivers active IAA to specific intracellular sites where enzymes catalyze its conjugation, hydrolysis, or degradation. Together, these interconnected processes create a dynamic regulatory network that coordinates auxin metabolism, transport, and compartmentalization to maintain auxin homeostasis and ensure precise control of plant growth and development.

Developing leaves undergo age-related physiological, hormonal and morphological changes that determine their adaptation plasticity towards stress conditions. We characterized waterlogging-induced leaf epinasty in tomato as one of these age-dependent cellular and hormonal responses. We quantified leaf angle dynamics and leaf physiology during waterlogging. Both old and young leaves rapidly bended down and showed an impaired photosynthesis and transpiration during waterlogging. Only young leaves partially recovered their posture and resumed photosynthesis after the waterlogging stress. Hormone analysis showed that young leaves have a higher capacity to convert the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) into the inactive conjugate malonyl-ACC, dampening ethylene synthesis during waterlogging. Petioles of older leaves showed a higher ethylene production capacity and were more sensitive towards waterlogging-induced stress. Ethylene stimulated cell elongation relatively more at the adaxial petiole side, by activating auxin accumulation in the petiole base of older leaves. Young leaves on the other hand, were less sensitive to waterlogging-induced ethylene and maintained their potential to transport auxins, leading to a milder epinastic response and a faster repositioning during reoxygenation. While the ethylene signaling inhibitor 1-methylcylcopropene inhibited epinasty, a 2,3,5-triiodobenzoic acid (TIBA) treatment enhanced epinastic bending, independent of waterlogging. We conclude that waterlogging activates intricate hormonal crosstalk between ethylene and auxin, controlled in an age-dependent way and leading to ontogenic differences in leaf epinasty and physiology.

The use of cell-free microbial culture filtrates (CF) as biostimulants is emerging as a safe and ecologically sound approach to improve crop performance while reducing anthropogenic pressure. However, the mechanisms underlying their biological activity remain poorly understood. We previously identified volatile organic compounds as major bioactive constituents of fungal CF. Here, we show that foliar application of cell-free CF derived from Trichoderma harzianum enhanced fruit yield, root growth, photosynthetic performance and agronomic water use efficiency in a commercial tomato cultivar grown in Mediterranean greenhouses under long-term water deficit conditions. To elucidate the biochemical and molecular bases of this phenomenon, we adopted an integrative approach to characterize plants grown under optimal and suboptimal irrigation conditions (OIC and SOIC, respectively) with or without the fungal CF treatment. Water deficit induced extensive changes in drought stress-related signaling molecules and in the leaf transcriptome, which accounted for many of the physiochemical differences recorded between OIC- and SOIC-grown plants. Notably, many of these changes were largely alleviated by foliar application of fungal CF to SOIC-grown plants, including those affecting the expression of approximately 50% of water deficit-responsive genes. These genes did not respond to CF in OIC-grown plants, indicating that the transcriptomic response to CF is strongly dependent on plant water status. Taken together, our results indicate that foliar application of fungal CF enhances tomato tolerance to long-term water deficit primarily by attenuating a substantial fraction of drought-induced metabolic and transcriptional responses rather than by inducing new ones, thereby reducing plant sensitivity to water stress.

Abscisic acid (ABA) acts as a key signalling molecule that mediates plant responses to environmental cues as well as plant growth and development. Stress-induced and developmental changes in ABA content trigger a myriad of post-transcriptional and transcriptional events. Yet, ABA-dependent transcriptional responses are context dependent, and their temporal dynamics in roots under non-stress conditions remain poorly resolved. In this study, we characterised the hallmarks of ABA signalling and responses in the poplar root transcriptome (Populus nigra L.). We disturbed ABA homeostasis by exogenous ABA treatments, and we combined time-resolved transcriptomics with unsupervised gene network analysis to identify ABA-activated and ABA-inactivated gene co-expression modules. Considering the temporal dynamics of transcriptional events, we predicted the primary targets of the ABA signal, characterised early responding ABA-dependent processes, identified hub genes and revealed their putative functional links. We demonstrated that the properties of a master ABA-activated module induced by exogenous treatments were preserved in the transcriptome response to osmotic stress, revealing a core gene set of ABA-dependent stress responses. Our work sheds light on ABA repression of gene expression, the reprogramming of metabolism and the leaf-senescence pathway. Based on current functional knowledge and phylogenetic information, including poplar-specific features, we proposed a working model of ABA action on root transcriptome in poplar that integrates master genes, key responsive processes, and their putative regulatory architecture.

Seed germination is a critical and highly regulated process that transitions a dormant seed to an actively growing seedling. This process plays a vital physiological role in regulating seedling establishment, plant growth, and development, while ecologically it shapes species distribution patterns, drives plant population dynamics, and influences ecosystem productivity. Seed germination is tightly controlled by various environmental and intrinsic factors, with phytohormones acting as primary mediators. Auxins, mainly indole-3-acetic acid (IAA), are involved in many aspects of plant growth and development. Accumulating evidence suggests that IAA modulates the balance between dormancy and germination similarly to abscisic acid (ABA) and gibberellins (GAs). In this mini-review, we summarize our current knowledge on the molecular mechanisms underlying the modulatory roles of IAA during seed germination. We specifically examine the crosstalk between IAA and other key phytohormones (ABA and GAs) that shape germination outcomes. Clarifying these interactions will enhance our understanding of the dormancy-germination switch and may offer practical methods to control germination timing in agriculture.

Potato is a globally important non-cereal crop in which infection with potato spindle tuber viroid (PSTVd) can cause stunted growth and significantly reduce tuber yield. We previously showed that PSTVd induces accumulation of the plant hormone jasmonic acid (JA) and alters antioxidant responses in potato plants. To clarify the role of JA in response to PSTVd, we analyzed disease development in transgenic JA-deficient opr3 and JA-insensitive coi1 lines compared to the wild-type. Transcriptomic analysis using RNA-Seq revealed that most genotype-specific differentially expressed genes (DEGs) in all comparisons were enriched in plant hormone signal transduction, plant-pathogen interaction, and MAPK signaling pathways, although the number of DEGs varied. These differences were confirmed by independent data from RT-qPCR, hormone, and hydrogen peroxide (H2O2) analyses. After PSTVd infection, opr3 plants showed enhanced JA signaling and increased abscisic acid (ABA) and auxin (AUX) content. In contrast, coi1 plants showed reduced ABA, AUX, and salicylic acid content. Both opr3 and coi1 plants showed reduced JA and H2O2 content and lower expression of defense-related genes, resulting in milder symptoms but increased viroid accumulation. In addition, treatment with methyl jasmonate alleviated symptoms in infected wild-type plants. Together, these results indicate a modulatory role for JA and JA signaling in basal immune responses and symptom development in the potato-PSTVd interaction.

Alongside biosynthesis and transport, inactivation regulates indole-3-acetic acid (IAA) concentration, a compound with numerous functions in plant development. The main inactive IAA metabolites are oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study IAA inactivation routes, we generated combinatorial Arabidopsis mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, and between DAOs and the main UGTs. In vivo [13C6]IAA feeding experiments traced exogenously applied IAA's metabolic fate, supporting the main IAA inactivation pathway where DAOs act downstream of GH3s. Results from these experiments also indicated UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data revealed that gh3oct may still retain some GH3 activity, explaining previously reported phenotypic inconsistencies. Our data additionally suggest that unidentified metabolic activities might play a role in IAA overproducing plants, and that premature downregulation of flowering time regulators like FLOWERING LOCUS C (FLC) likely underlies early flowering of gh3oct and gh3oct dao1-6 plants.

Abstract Auxin impacts on nearly every aspect of plant growth and development. Its exogenous application therefore results in pleiotropic growth responses. Exploiting this activity for plant propagation requires avoiding or minimizing such off-target effects and is generally achieved as a trade-off between toxicity and organogenetic efficacity. We recently identified the compound HYSPARIN (HYS) with potent, and uniquely selective adventitious root inductive activity. Unlike other root-inducing compounds, HYS preferentially activates auxin responses in the shoot via an unknown mechanism. Here, we show that HYS acts as a shoot-specific proauxin. Rather than acting through auxin homeostasis, we found that HYS is hydrolysed i n planta independently of ILR1/ILL amidohydrolases to release the synthetic auxin MCPA. Structure–activity relationship analysis confirmed a strong dependence on its MCPA moiety for activating auxin responses, and identified its promoiety as a determinant of shoot-specificity and activity. Selective application of MCPA also potently induces AR is consistent with a model in which HYS metabolism produces a spatially restricted, AR inductive auxin signal. The activation mechanism of HYS thus provides a conceptual framework for tissue-specific metabolic delivery of auxin and may enable the programmable delivery of other xenobiotics in plants.

Previous studies have highlighted a positive role of ethylene in regulating in vitro shoot regeneration in the woody species Solanum betaceum Cav. (tamarillo). However, the involvement of ethylene in the indirect somatic embryogenesis (SE) process of tamarillo remains insufficiently understood. The main objective of this study was, therefore, to elucidate how ethylene influences embryogenic callus induction and somatic embryo development in tamarillo by chemically modulating its biosynthesis and perception. The results demonstrated that ethylene is essential for the induction of embryogenic callus, whereas inhibition of its biosynthesis by aminoethoxyvinylglycine (AVG) enhanced somatic embryo differentiation. Leaf explants treated with AVG exhibited delayed cell dedifferentiation and impaired acquisition of totipotency, along with associated changes, including higher cytokinin (CK) levels and upregulation of the positive regulator of CK responses, ARR1. During the development of somatic embryos, AVG treatments promoted somatic embryo differentiation, whereas application of the ethylene-releasing compound, ethephon, negatively affected both embryo formation and differentiation by downregulating the expression of the SE-related gene BBM2 and reducing CK levels. Our findings indicate that a rapid increase in endogenous 1-aminocyclopropane-1-carboxylic acid (ACC) levels, together with increased SERK1 and BBM2 expression, during the early weeks of SE, are associated with cell dedifferentiation and further acquisition of embryogenic competence, while lower ACC levels are required for subsequent embryo initiation. Overall, this study provides novel insights into the molecular and metabolic mechanisms regulated by ethylene during the SE process, with potential for improving in vitro regeneration systems in tamarillo and other woody species. Ethylene is required for auxin-induced embryogenic callus formation from tamarillo leaf explants but negatively affects subsequent somatic embryo development under hormone-free conditions.

Acidic phytohormones, such as abscisates, auxins, jasmonates, and salicylic acid, are low-abundance signalling molecules in plants. A high-throughput workflow coupling reverse phase based miniaturised solid phase extraction (RP-μSPE) with ultra-high performance supercritical fluid chromatography tandem mass spectrometry method (SFC-MS/MS) was established for their profiling, introducing complementary selectivity in sample clean-up and chromatographic separation. The SFC-MS/MS method was developed through stationary phase selection, design of experiment screening, and optimisation of chromatographic conditions, ion-source parameters, make-up solvent, injection volume, and gradient conditions. The Viridis HSS C18 SB column was selected among five stationary phases of different chemistries, supercritical CO2 and methanol as a co-solvent, containing 0.1% ammonia and 3% water, were employed as the mobile phase. Direct injection of 5 μL μSPE eluate enabled satisfactory peak shapes without an evaporation step. Methanol was used as the make-up solvent at 0.25 mL/min. The workflow was validated, providing accuracies within 85-115%, precision of 0.2-14.9%, matrix effects of 87-109%, limits of detection 2.5-250 fmol, linear ranges spanning 3-4 orders of magnitude, R2 0.9946-0.9999, and carry-over not exceeding 20%, for 19 acidic phytohormones passing the validation criteria. Compared to the routine RP-LC workflow, the SFC approach reduced chromatographic run time from 19 to 11 min, offered complementary selectivity, and decreased matrix effects. However, in SFC, the detection sensitivity was about one order reduced, two compounds were excluded from the method due to strong carry-over and one due to insufficient retention relative to LC. The workflow applicability was demonstrated by time-course profiling in wounded Arabidopsis thaliana leaves.

Seedlings are highly vulnerable to various stresses due to their underdeveloped root and shoot systems. Climatic changes and abiotic stresses further hinder seedling emergence, while the excessive use of inorganic inputs harms soil health. Organically derived biostimulants, such as smoke-water, vermicompost leachate, and seaweed extract, offer promising alternatives. While their individual effects are well-documented, little is known about their combined role in seedling development. This study evaluated the synergistic effects of smoke-water (1:2000 v/v), vermicompost leachate (1:20 v/v), and Kelpak (0.5

Together with biosynthesis and transport, inactivation regulates the concentration of indole-3-acetic acid (IAA), a key auxinic compound with a myriad of functions in plant development. Main inactive IAA metabolites are categorised into oxidised forms and ester- or amide-linked conjugates. DIOXYGENASE FOR AUXIN OXIDATION1 (DAO1) and DAO2, 2-oxoglutarate and iron-dependent dioxygenases, contribute to IAA oxidative inactivation in collaboration with group II GRETCHEN HAGEN3 (GH3) IAA-amido synthetases, while a group of UDP-glycosyltransferases (UGTs) conjugate IAA to sugars. To study the IAA inactivation routes, we generated combinatorial mutants between all group II GH3s (gh3oct) and DAO1 or DAO2, as well as between the DAOs and main UGTs. In vivo [13C6]IAA feeding experiments traced the metabolic fate of the exogenously applied IAA, supporting the main IAA inactivation pathway, in which DAO acts downstream of GH3s. They also indicated that UGT-mediated IAA glycosylation is more important than previously assumed for modulating IAA levels and plant development. Our metabolic and transcriptomic data further revealed that gh3oct may still produce some GH3 activity, explaining previous reported phenotypic inconsistencies. Our data additionally suggest that other not yet identified metabolic activities might play a role in IAA overproducing plants, and that the premature downregulation of flowering time integrators like FLOWERING LOCUS C (FLC) likely underlies the early flowering of gh3oct and gh3oct dao1 plants. ### Competing Interest Statement The authors have declared no competing interest. Knut and Alice Wallenberg Foundation Knut and Alice Wallenberg Foundation, WIFORCE VINNOVA, https://ror.org/01kd5m353 Swedish Research Council, https://ror.org/03zttf063 Kempestiftelserna, JCK-1811, JCK-1111 University of Nottingham Royal Society, RGS\_R1\_191323 European Research Council, 101166880 Agencia Estatal de Investigación, RYC2021-030895-I, PID2023-147737NA-I00

Modern agriculture’s reliance on synthetic agrochemicals has raised concerns over environmental sustainability, prompting exploration of eco-friendly alternatives such as natural biostimulants. This study evaluates the individual and combined effects of three biostimulants viz., smoke-water, seaweed extract (Kelpak), and vermicompost leachate on maize seed germination, seedling growth, hydrolytic enzymatic activity, and phytohormone profiles under light and dark conditions. Maize seeds were treated with various concentrations of each biostimulant and a combined formulation (CBS), and germination performance, α-amylase activity, and levels of selected phytohormones were assessed. Results showed that vermicompost leachate and the combined biostimulant treatment significantly enhanced root length and seedling vigour index, particularly under dark conditions. CBS treatment also elevated α-amylase activity under both light and dark regimes, indicating improved metabolic activation during germination. Phytohormone analysis revealed that CBS reduced levels of auxin, its conjugates, and abscisic acid under light, suggesting biostimulant-mediated hormonal modulation. No significant hormonal changes were observed under dark conditions, except for decreased oxIAA (2-oxindole-3-acetic acid) in CBS-treated seedlings. These findings demonstrate that biostimulant combinations can exert additive effects, enhancing early growth and metabolic activity while modulating hormone levels in maize seedlings. The study highlights the potential of integrated biostimulant strategies to support sustainable crop production.

The transcription factor WIP2/NO TRANSMITTING TRACT (WIP2/NTT) belongs to the WIP zinc finger family. Loss of WIP/NTT function in Arabidopsis thaliana causes alterations in specific tissues in the gynoecium. It also impairs root development, but only when combined with the loss of WIP4 and WIP5 function, due to redundancy. Certain mutant loss-of-function phenotypes can be recovered by cytokinin application, NTT interacts with cytokinin signaling components, and the phenotypes displayed by plants with increased WIP2/NTT expression also suggest a possible interaction with this pathway. Therefore, the objective of this study was to investigate the relationship between WIP2/NTT and the cytokinin pathway. To overcome the issue of genetic redundancy, we used a commonly used inducible system. We found that WIP2/NTT induction alters cytokinin levels and signaling in a tissue-specific manner, as shown by cytokinin content measurements and TCSn::GFP reporter analysis. Transcriptome analyses revealed candidate target genes related to the cytokinin pathway. Yeast one-hybrid and transactivation assays demonstrated direct NTT binding to regulatory regions of the cytokinin genes ISOPENTENYL TRANSFERASE 5 (IPT5), ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), and CYTOKININ OXIDASE/DEHYDROGENASE 7 (CKX7) involved in cytokinin biosynthesis, signaling, and degradation, respectively. Moreover, immunolocalization assays revealed that cytokinin distribution was altered in loss of function mutants and after NTT induction. The results of this work indicate that WIP2/NTT modulates cytokinin homeostasis.

Each of the 4 different cytokinin (CK) base forms, trans-zeatin (tZ), isopentenyladenine (iP), dihydrozeatin (DHZ), and cis-zeatin (cZ) has distinct chemical metabolism and affinity to the CK Histidine Kinase (CHK) receptors. However, it remains unclear how the specific biochemical features of each form, such as receptor specificity or metabolic differences, drive distinct tissue-specific physiological output in response to application of these CK bases. Here, we show that CK receptor preference and metabolic persistence together shape isoform-specific CK signaling strength, including tissue-dependent hormone responses in Arabidopsis leaf versus root assays. Physiological, genetic, and multi-omics integration was used to show that tZ and iP anti-senescence activity is matched by DHZ through a distinct receptor metabolic mechanism. DHZ requires Arabidopsis Histidine Kinase 3 (AHK3) signaling to be fully effective in a leaf dark-induced senescence (DIS) assay and where it overcomes its lower receptor affinity through higher metabolic persistence, accumulating at levels ∼2.5-fold above tZ and iP early in a senescence time course. Together, these findings provide a framework for integration of receptor preference and metabolic stability to determine CK isoform activity.

Land plants have co-evolved with microorganisms since their transition to a terrestrial habitat, around 500 million years ago. In angiosperms, salicylic acid (SA) activates plant immunity against hemibiotrophic pathogens through TGACG-motif-binding (TGA) transcription factors, which bind to the promoter of SA-responsive loci, such as pathogenesis-related (PR) genes, to enforce plant immunity. While those mechanisms are well-known in flowering plants, our understanding in bryophytes remains limited, as genetic evidences for the role of SA during plant immunity are still missing. Here, we explore the interaction between Marchantia polymorpha and the bacterium Pseudomonas syringae to gain insights into the evolutionary immune function of SA during bryophyte-pathogen interactions. We combined transcriptomic profiling of P. syringae-infected Marchantia with the generation of SA-deficient plants in this liverwort by overexpressing the bacterial NahG gene, a SA-degrading enzyme. Our results indicate that the P. syringae-induced transcriptional footprint is enriched in SA-responsive genes and that SA-deficient Marchantia NahG plants are compromised in immune responses against P. syringae. We show that the unique MpTGA is essential for controlling resistance against Pseudomonas. Further transcriptional analyses into the coregulatory network controlled by SA and MpTGA indicate that an SA/MpTGA module activates plant defence responses through a variety of MpPRs, enriched in the regulation of class III of secretory peroxidases belonging to the MpPR9 subfamily during the early defensive response against P. syringae. Altogether, our data demonstrate the functional conservation of SA as an immune hormone and underpin the existence of a SA/MpTGA-regulated transcriptional cluster driving resistance against Pseudomonas in Marchantia.

This protocol describes the extraction of low-molecular-weight (LMW) polar and semi-polar compounds from mouse serum and brain tissue for quantitation by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Proteins are precipitated by cold organic solvent (1:1 acetonitrile:methanol + 0.1% formic acid, pre-chilled to −20 °C). The clarified extract is sequentially filtered through a 0.22 µm filter and then a 30 kDa molecular-weight cut-off (MWCO) membrane to remove residual particulates and macromolecules before analysis. Stable isotope-labeled internal standards may be spiked into the extraction solvent for all study samples. Serum and brain tissue are processed in parallel from the same animal at the terminal time point.