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.

Cytokinins (CKs) delay dark-induced senescence, but how they tune photosynthetic function in darkness remains unclear. We investigated the effects of classical aromatic CK benzylaminopurine and CK-derived compound 1-(2-methoxyethyl)-3-(1,2,3-thiadiazol-5-yl)urea on detached Arabidopsis leaves at different time points during dark incubation. Notably, while both compounds mitigated the progression of senescence, they unexpectedly downregulated Photosystem II (PSII) photochemistry during the early stages of dark incubation. Despite this downregulation, CK-treated leaves preserved functional photochemistry and substantially delayed Chl degradation at later time points. Transcriptomic analysis at the early stage of darkening revealed that a significant portion of CK-responsive genes is associated with photosynthesis, PSII function, and light sensing, including red-light signaling pathways. Differential proteomics further supported a CK-induced shift to a metabolically suppressed state. The early PSII downregulation required phytochrome B, indicating a CK-phyB module that places darkened leaves into a reversible 'standby' mode, limiting energy demand and enabling rapid, safer recovery of photosynthesis upon re-illumination. At the same time, it may also protect the photosynthetic apparatus against photo-oxidative damage during the transition from dark- to light-adapted states.

Quantifying plant hormones (phytohormones) is essential for understanding plant growth and development. Among the phytohormones, cytokinins are crucial for plant growth, and have also emerged as interkingdom signaling molecules between roots and root-associated microbes. However, existing methods do not allow for non-invasive, in-situ hormone level determination in these interactions. Here we present a label-free electrochemical impedance spectroscopy (EIS) immunosensor to detect the cytokinin molecule, isopentenyladenine (iP) by immobilizing a monoclonal antibody on a self-assembled monolayer (SAM)-modified screen-printed gold electrode (SPAuE). For small molecule measurements using such antibody-based sensors, signal drift in EIS measurements is very common leading to false positive or false negative signals. To address this issue of signal drift, we incubated the antibody-modified electrodes in nitrogen-purged, deaerated phosphate-buffered saline (N-PBS), which resulted in more reproducible and accurate measurements. After incubating the immunosensor with iP in N-PBS, it had a linear response to iP in the concentration range 10-1000 nM with minimal interference towards common small molecules co-existing in plant samples. Finally, the immunosensor was incubated with spiked concentrations of iP in plant root exudate where a linear response to iP was found in the concentration range 100-1000 nM. The developed immunosensor has the potential to quantify other cytokinin molecules with minor modifications.

Previously, we showed that altered sulfite homeostasis led to higher water loss in Sulfite oxidase RNA interference (SO Ri) plants than in Arabidopsis wild-type (WT) after sulfite infiltration into rosette leaves. In contrast, SO overexpression (OE) and adenosine-5'-phosphosulfate reductase (apr2) KO resulted in lower water loss than in WT. Accordingly, sulfite homeostasis and drought tolerance under prolonged drought in Arabidopsis thaliana and tomato (Rheinlands Ruhm) were investigated. SO Ri mutants displayed the most severe wilting and water loss, accompanied by sulfite accumulation due to reduced SO expression. In contrast, SO OE, apr2 KO, apr2 KO/SO OE, and gene-edited lines, including SiR OEC2+SO OEC2 and SiR OEC4/SO OE maintained higher relative water content (RWC) by enhancing sulfite oxidation and reduction. Elevated sulfite in SO Ri mutants acted as an antagonist to abscisic acid (ABA), as despite accumulating higher ABA than all other genotypes, SO Ri plants displayed the lowest RWC, consistent with sulfite suppressing ABA signal transduction through impaired ABA perception (PYL5) and enhanced ABA negative feedback (HAI2). SO and SiR overexpression also increased hydrogen sulfide and cysteine levels, contributing to drought resilience. Tomato lines also reflected these patterns in SO OE, SiR OE, and SiR OE/SO OE, showing reduced sulfite and improved RWC compared to WT. Overall, coordinated regulation of SO, SiR, and APR2 maintains sulfite homeostasis, mitigating drought-induced damage and enhancing water retention across species by directly limiting sulfite accumulation and relieving sulfite-driven antagonism of ABA signal transduction.

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.

Brassinosteroids (BRs) are chemically diverse plant steroid hormones produced via a branched biosynthetic pathway. The potent BR brassinolide is sensed by the membrane receptor kinase BRI1 and a SERK co-receptor, but the physiological functions of other abundant BRs remain to be characterized. Here we present quantitative binding kinetics for 4 Arabidopsis thaliana BR receptors and 15 BRs, which define the key chemical features required for high-affinity receptor binding, ligand positioning and co-receptor recognition. BRI1, BRL1 and BRL3 share overlapping ligand preferences, whereas BRL2 binds C28 BRs with moderate affinity. Structural analyses of BR-bound BRI1 and BRL3 ectodomains combined with extensive in vitro and in vivo mutagenesis studies reveal a high structural plasticity of the hormone-binding pocket. Functional assays using structure-based BR agonists and antagonists uncover that BR receptor-co-receptor signalling complexes can recognize chemically diverse BRs, introducing an additional, intriguing layer of BR signalling regulation.

Enhanced Apiaceae germination performance by seed priming involves promoting pre-germination growth of the underdeveloped (small) embryos, reduction in hormone contents, and priming with abscisic acid (ABA) improved ageing resilience. Different seed priming technologies are used to improve germination performance and seedling vigour of vegetable crops. Daucus carota (carrot), Pastinaca sativa (parsnip), and other Apiaceae produce morphologically dormant single-seeded fruit halves (mericarps) as dispersal units. In mature mericarps, the underdeveloped (small) embryo is embedded in abundant endosperm tissue, and pre-germination embryo growth to a critical embryo:seed (E:S) length ratio is a requirement for the completion of germination by radicle emergence. We investigated how hydropriming and additive priming with gibberellins (GA), abscisic acid (ABA), and gas plasma-activated water (GPAW) affected carrot and parsnip mericarp germination and ageing sensitivity accessed using a wet ageing assay (80

Different cytokinin (CK) forms have distinct receptor affinities and metabolic rates, as seen in previous work with trans -Zeatin ( t Z), isopentenyladenine (iP), dihydrozeatin (DHZ), and cis -Zeatin ( c Z). However, it remains unclear how specific biochemical features of each form drives distinct tissue-specific physiological hormone output in response to application of these CK bases. Likewise, highly abundant N-glucoside CKs have also recently been attributed with having tissue-specific activities, yet their contribution to CK signaling output remains unclear. Here, we show that CK receptor preference and metabolic persistence together shape isoform-specific CK signaling strength, including tissue-dependent hormone responses in leaf versus root assays. We used physiological, genetic, and multi-omics integration in Arabidopsis to show that t Z and iP anti-senescence activity is matched by DHZ through a distinct receptor metabolic mechanism. DHZ requires AHK3→ARR2 signaling to be fully effective in Dark Induced Senescence (DIS) assay and 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. Second, we demonstrate that N-glucosides, in a ratio-dependent manner, can act as modulators of CK response intensity. t Z N-glucoside co-applied with its base isoform t Z reduces CK signaling output by up to 41% as seen by pTCS::LUC in both protoplast transient expression and whole-leaf assays. Together, these findings provide a framework of how integration of receptor preference and metabolic stability determines CK isoform activity, in a model where abundant N-glucosides can modulate active CK signaling output. ### Competing Interest Statement The authors have declared no competing interest. All data are incorporated into this article and its online supplementary material. Customized R scripts are in the GitHub Repository () U.S. National Science Foundation, 2033337 Alabama Agriculture Experiment Station, ALA021-1-19083 European Regional Development, CZ.02.01.01/00/23_020/0008497

A parthenocarpic fruit mutant of prickly pear was isolated, revealing the role of GAs in parthenocarpic fruit development which is controlled by the GID-GA20ox/GA2ox genetic system modulating GA biosynthesis/regulation. We explored the intricate dynamics of parthenocarpic fruit development in prickly pear Opuntia ficus-indica (Cactaceae) through the investigation of fruits of the Beer Sheva1 (BS1) a parthenocarpic mutant and its revertant non-parthenocarpic stems. BS1 fruits, characterized by parthenocarpy and enlarged unfertilized ovules, provide a unique model for investigating the regulatory mechanisms underlying fruit development in prickly pear. We hypothesized that elevated levels of gibberellins (GAs) in BS1 ovaries induce parthenocarpic fruit development. By integrating different approaches, including GA quantification and expression analysis of ovaries from BS1 and revertant flowers, we elucidated the pivotal role of biosynthetic, catabolic, and regulatory GA genes in orchestrating ovule development. Notably, our investigation revealed a complex interplay between GA biosynthesis and catabolic genes, particularly GID1, GA20ox, and GA2ox, which significantly influenced GA levels in BS1 ovaries. Quantification of endogenous GAs confirmed higher levels of bioactive GA1, GA3, and GA4 in BS1 compared to revertant ovules, indicating the central role of GAs in parthenocarpy. Furthermore, application of the GA inhibitor paclobutrazol (PBZ) to BS1 flower buds resulted in the reversion of BS1 fruits to the progenitor phenotype containing viable seeds, thereby validating the critical involvement of GAs in seed development. High-throughput RNA-sequencing analysis identified a total of 7717 differentially expressed genes (DEGs) in BS1, among them GA-related genes. Overall, our findings shed light on the complex hormonal regulatory network governing parthenocarpic fruit development in prickly pear, paving the way for future studies aiming at understanding ovule development and development of commercially desirable seedless fruits.

Familial dysautonomia is a debilitating congenital neurodegenerative disorder with no causative therapy. It is caused by a homozygous mutation in ELP1 gene, resulting in the production of the transcript lacking exon 20. The compounds studied as potential treatments include the clinical candidate kinetin, a plant hormone from the cytokinin family. We explored the relationship between the structure of a set of kinetin derivatives (N = 72) and their ability to correct aberrant splicing of the ELP1 gene. Active compounds can be obtained by the substitution of the purine ring with chlorine and fluorine at the C2 atom, with a small alkyl group at the N7 atom, or with diverse groups at the C8 atom. On the other hand, a substitution at the N3 or N9 atoms resulted in a loss of activity. We successfully tested a hypothesis inspired by the remarkable tolerance of the position C8 to substitution, postulating that the imidazole of the purine moiety is not required for the activity. We also evaluated the activity of phytohormones from other families, but none of them corrected ELP1 mRNA aberrant splicing. A panel of in vitro ADME assays, including evaluation of transport across model barriers, stability in plasma and in the presence of liver microsomal fraction as well as plasma protein binding, was used for an initial estimation of the potential bioavailability of the active compounds. Finally, a RNA-seq data suggest that 8-aminokinetin modulates expression spliceosome components.

While cytokinin (CK) can delay natural leaf senescence, its effects on abiotic stress accelerated leaf senescence are less studied. Here we show N-conjugated trans-zeatin CK forms (tZ7G and tZ9G, or tZNGs) have the ability to delay salt stress senescence. Using a modified dark-induced senescence bioassay with Arabidopsis leaves, exogenous salt treatment accelerated leaf senescence as measured by lower photosystem II efficiency (Fv/Fm) and chlorophyll content. tZNGs were able to delay these parameters at concentrations as low as 10 nM similar to tZ, indicating that tZ7G and tZ9G can function in delaying salt accelerated senescence (SAS). To better understand physiological effects regulating tZNG delay of senescence, transcriptomics, proteomics, as well as CK measurements were examined. Salt treatment has strong transcriptome and proteome effects in accelerating senescence and reducing overall CK levels. Exogenous CK treatments could be quickly detected from changes seen in endogenous CK measurements, where each CK has a distinct profile contributing to transcript/protein alterations. Interestingly, transcriptomics show tZNGs are primarily responsive at later stages of salt senescence, in contrast to an immediate and continual response of tZ treatment. Known CK-regulated genes are induced by tZNGs and tZ, as corroborated by ARR:GUS reporter lines. Differences between tZNGs and tZ DEGs were revealed by WGCNA that included salt and CK specifically gene modules. In contrast, proteomic analysis revealed unique, but similar numbers of tZNG compared to tZ DAPs across senescence. GO term analysis of tZNG DEGs and DAPs showed enrichment of senescence, chloroplast, and CK signaling. Together this indicates tZNGs function as active CK forms in delaying salt accelerated leaf senescence.

Elevated temperatures caused by climate change threaten potato production. To understand heat stress adaptations and variety-specific responses, plants of a susceptible (Cecile) and a tolerant cultivar (Solara) were exposed to elevated temperatures (30/28 °C) for 21 days at tuberization stage. Phenotypic, physiological, transcriptional and metabolic changes were analyzed in comparison to ambient temperatures (21/19 °C). Heat stress caused shoot elongation and tuber weight loss, which were more pronounced in Cecile. Transcriptome analysis of leaf samples revealed a stronger decrease of photosynthesis-associated genes in the sensitive cultivar Cecile, which was associated with decreased chlorophyll fluorescence and an early senescence. These effects correlated with strongly elevated levels of salicylic acid and ethylene. In contrast, Solara showed delayed senescence and a higher expression of sugar and amino acid transporters suggesting an adaptive mechanism to maintain carbohydrate and amino acid allocation. The expression of known tuberization regulators including SP6A, exhibited a similar response to heat in both varieties, with decreasing expression of SP6A. Solara exhibited a constitutively higher expression of PEBP14/15 and MADS13, which potentially promote tuberization and may support tuber growth under heat. Regardless of variety, a few genes, such as HSP20 and HSP70, were induced by heat and may serve as heat stress marker genes. Altogether, the results indicate that delayed senescence, stable photosynthesis, efficient assimilate translocation, and differential regulation of tuberization pathways contribute to heat tolerance in Solara. These insights improve our understanding of the molecular basis of heat resilience and provide potential targets for breeding climate-resilient potato varieties.

Cytokinins, known as the compounds of natural origin, play a significant regulatory role in the senescence in plants. Several of them, such as 6-furfurylaminopurine (kinetin), also showed beneficial effects on human skin cells. We focused on structurally similar cytokinin 6-(4-hydroxybenzylamino)purine, p-topolin (pT), and its water-soluble mesylate salt (pTM). Newly prepared salt is 60 000 times more soluble in water than pT. Both compounds are UVA photostable, non-toxic and non-phototoxic. We also explored UVA protective effect on human fibroblasts (NHDF), keratinocytes (HaCaT) and porcine ear skin. Since the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway regulates various genes in regulation of oxidative stress, we focused on effects of pT and pTM in this manner. Both compounds increased translocation of Nrf2 transcription factor to the nucleus in NHDF and stimulated the accumulation of Bach1, a Nrf2 transcription regulator in HaCaT. Studied compounds also significantly reduced UVA-induced UPE (ultra-weak photon emission) related to reactive oxygen species (ROS) formation in skin cells and porcine ear skin ex vivo, in which pTM was found to have higher protective effect compared to pT.

Adenosine undergoes ATP-dependent phosphorylation catalyzed by adenosine kinase (ADK). In plants, ADK also phosphorylates cytokinin ribosides, transport forms of the hormone. Here, we investigated the substrate preferences, oligomeric states, and structures of ADKs from moss (Physcomitrella patens) and maize (Zea mays) alongside metabolomic and phenotypic analyses. We showed that dexamethasone-inducible ZmADK overexpressor lines in Arabidopsis can benefit from a higher number of lateral roots and larger root areas under nitrogen starvation. We discovered that maize and moss enzymes can form dimers upon increasing protein concentration, setting them apart from the monomeric human and protozoal ADKs. Structural and kinetic analyses revealed a catalytically inactive unique dimer. Within the dimer, both active sites are mutually blocked. The activity of moss ADKs, exhibiting a higher propensity to dimerize, was 10-fold lower compared with maize ADKs. Two monomeric structures in a ternary complex highlight the characteristic transition from an open to a closed state upon substrate binding. This suggests that the oligomeric state switch can modulate the activity of moss ADKs and probably other plant ADKs. Moreover, dimer association represents a novel negative feedback mechanism, helping to maintain steady levels of adenosine and AMP.

Root-knot nematodes ( Meloidogyne spp.) are obligatory plant root parasites whose effector proteins play a critical role in suppressing plant immunity. However, the effectors direct host targets and underlying molecular mechanisms remain poorly understood. Using TurboID-mediated proximity labeling in tomato ( Solanum lycopersicum ) hairy roots, we identified an interaction between the nematode effector Mj-MSP18 and the tomato BRASSINOSTEROID-SIGNALING KINASE 7 (Sl-BSK7). Yeast two-hybrid (Y2H) assays confirmed that this interaction is conserved in Arabidopsis thaliana and Nicotiana benthamiana . Additionally, yeast three-hybrid and luciferase complementation assays demonstrated that Mj-MSP18 disrupts the interaction between BSK7/8 and FLS2 in both yeast and in planta . Given that the BSK7/8–FLS2 interaction is essential for flg22-induced pattern-triggered immunity (PTI), this disruption likely accounts for the suppression of reactive oxygen species (ROS) production and callose deposition observed upon transient expression of Mj-MSP18 in flg22-treated N. benthamiana leaves. Correspondingly, Arabidopsis and tomato bsk7 mutants exhibited increased susceptibility to root-knot nematode infection. Furthermore, RNA-seq analysis of tomato hairy roots expressing Mj-MSP18 revealed extensive transcriptional reprogramming, including the downregulation of defence-related genes and hydrogen peroxide response pathways. In addition, Y2H screening identified Sl-MYB and Sl-MYC2 as additional interactors, linking Mj-MSP18 to phytohormone biosynthesis, particularly the brassinosteroid (BR) and salicylic acid (SA) pathways, as validated by targeted metabolite analysis. The conservation of Mj-MSP18 across Meloidogyne species suggests a broadly conserved mechanism for host immune suppression and phytohormone modulation. ### Competing Interest Statement The authors have declared no competing interest. Special Research Fund of Ghent University Research Foundation - Flanders, https://ror.org/03qtxy027, G045921N Czech Science Foundation, 22-17435S China Scholarship Council, https://ror.org/04atp4p48