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.

Leaf senescence is often characterized by visible yellowing, reflecting the degradation of photosynthetic pigments and the functional deterioration of photosynthetic complexes, marking a shift from photosynthetic maintenance to nutrient remobilization. Cytokinins are key modulators in this process. This review examines cytokinin action in mature Arabidopsis leaves from the perspective of chloroplast functional lifetime, focusing on how long-distance supply, local metabolism and candidate transport routes may shape cytokinin availability and action during leaf senescence. Cytokinin signaling is further considered in relation to nitrogen status, because chloroplasts are both sites of photosynthetic carbon assimilation and major reservoirs of remobilizable nitrogen. As senescence progresses, chloroplasts are not merely passive targets of degradation. Declining photosynthetic function can generate retrograde signals, alter reactive oxygen species homeostasis, and promote transcriptional programs that accelerate dismantling of the photosynthetic apparatus. Within this framework, cytokinins help preserve chlorophyll content, photosynthetic complexes, Photosystem II function, and chloroplast-related gene expression while buffering redox- and light-dependent feedbacks. Cytokinin action in mature leaves is therefore discussed here mainly in the context of spatiotemporal regulation of chloroplast functional lifetime. Cytokinin availability, nitrogen status, and light-dependent feedback from the photosynthetic apparatus jointly influence when photosynthetic maintenance gives way to chloroplast dismantling and nutrient remobilization. This perspective also reconciles recent evidence from detached Arabidopsis leaves that cytokinins can transiently repress PSII photochemistry under prolonged darkness while delaying subsequent chlorophyll loss, adding another layer to cytokinin-mediated protection during early stages of senescence.

This study compares the ability of the cytokinin (CK) trans-zeatin (tZ) and the CK sugar conjugate 6-(3-methoxybenzylamino)purine-9-arabinoside (BAPA) to induce resistance against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Treatment with either tZ or BAPA significantly reduced bacterial growth after a later infection. This chemically induced resistance (IR) required the CK receptor AHK3, highlighting its critical role in mediating resistance by tZ and BAPA. This is remarkable as these compounds show either high or no affinity for this CK receptor, respectively. Surprisingly, tZ, but not BAPA, induced the expression of CK response genes, including ARR5, suggesting divergent mechanisms of action. Resistance caused by both compounds was abolished in the npr1 mutant, underpinning the functional relevance of the salicylic acid (SA) signalling pathway. Transcriptomic analysis showed that both BAPA and tZ triggered the expression of distinct sets of genes associated with SA and reactive oxygen species (ROS) but not with jasmonic acid (JA) signalling. BAPA and, to a lesser extent, also tZ activated pattern-triggered immunity (PTI) signalling genes, including genes responsible for PTI signal amplification (PREPIP2) and pathogen-associated molecular pattern (PAMP) signalling (PH1, IDL6). This supported the hypothesis that the PTI pathway mediates the protective effect. Similarities and differences of chemically triggered IR by tZ and BAPA, as well as their potential for application, are discussed.

Botrytis cinerea, a highly successful necrotrophic fungal plant pathogen, demonstrates a remarkably broad host range, yet the precise factors governing its host specificity and its ability to coexist non-pathogenically with plants remain unclear. This study systematically assessed host preference in a series of B. cinerea agricultural isolates, revealing a degree of host preference and reduced necrotrophy on "non-hosts". Examining which factors affect these observed host preferences, we identified leaf structure and cytokinin (CK) content as key factors in host recognition. Investigating non-pathogenic B. cinerea-plant interactions, we found that B. cinerea can induce cellular immunity and broad-spectrum disease resistance, with some isolates promoting plant growth and productivity. B. cinerea induced immunity primarily required the salicylic acid (SA) and ethylene (ET) pathways in the host plant. Our findings provide empirical insights into B. cinerea's host adaptation and lifestyle versatility, highlighting a beneficial aspect of its interaction with plants, which could inform environmentally friendly disease control strategies.

Cytokinin ribosides are major mobile and precursor forms of cytokinins, plant hormones whose transport and subcellular distribution shape developmental and stress responses. Here, we identify Arabidopsis thaliana EQUILIBRATIVE NUCLEOSIDE TRANSPORTER1 (ENT1) as a tonoplast-localized cytokinin riboside transporter. Tissue-specific subcellular analysis under native regulatory elements localized ENT1 predominantly to the tonoplast of root epidermal and lateral root cap cells, where it gates intracellular cytokinin riboside availability. Accordingly, ENT1 overexpression enhanced cytokinin riboside sensitivity and signalling, whereas loss of ENT1 altered adenosine metabolism and disrupted cytokinin homeostasis, leading to the accumulation of multiple zeatin-type cytokinins. ENT1-dependent cytokinin riboside compartmentalization was required for beneficial microbe-induced protection, as ent1 mutants failed to acquire protection against the fungal pathogen Botrytis cinerea and the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. These findings reveal a vacuolar gatekeeping mechanism that controls intracellular cytokinin riboside availability and links hormone compartmentalization to beneficial microbe-dependent plant defence.

Cytokinins (CKs) regulate numerous biological processes in plants, and are involved in plant growth and development as well as responses to biotic and abiotic stresses. Although CKs have been reported to prime plant immunity, thus far, research on CK hormopriming has focused on classical synthetic CKs such as kinetin or 6-benzylaminopurine (BAP). Here, we show that synthetic aromatic CK derivatives—specifically, 3‑methoxy-6-benzylaminopurine-9-arabinosides—effectively induce immune responses and promote resistance to Botrytis cinerea in tomato (Solanum lycopersicum) primarily via a salicylic acid (SA)-dependent mechanism. Additionally, like 6-BAP, these compounds directly inhibit B. cinerea. Transcriptomic analyses of B. cinerea treated with CK arabinosides revealed significant changes in expressed gene profiles, notably in pathways associated with sugar metabolism. Unlike classical CKs, CK arabinosides do not alleviate growth inhibition under sugar-limited conditions, contributing to their stronger antifungal effects. Furthermore, CK arabinosides induce tomato immune responses more robustly than classical CKs, yet without activating canonical cytokinin signaling pathways, likely owing to their exceptional chemical stability which prevents release of the free active CK base, and due to their inability to directly bind CK receptors. Altogether, our findings suggest that CK arabinosides act via a dual mechanism—immune priming and direct pathogen inhibition—and hold promise as next-generation hormopriming agents for sustainable crop protection.

Ribosylated forms of the plant hormones cytokinins (CKs) are the dominant CK species translocated over long distances. The irreplaceable role of root-to-shoot translocated trans-zeatin riboside in the mediation of shoot development implies the existence of a yet-uncharacterized CK riboside-specific membrane transport system. In this work, we report significant differences in the kinetics of the membrane transport of CK nucleobases and ribosides and the overall affinity of membrane-bound carriers towards the two CK forms. We further characterize the membrane transport of CK nucleobases and ribosides mediated by Arabidopsis EQULIBRATIVE NUCLEOSIDE TRANSPORTER 3 (AtENT3) in tobacco BY-2 cells. Combining experimental data with computational modelling, we show that residues Tyr61 and Asp129, which are conserved among plant ENTs but not among ENTs from other species, are necessary for CK binding and that their mutation abolishes the ability of AtENT3 to transport CKs. Finally, we show that changes in AtENT3 have different effects on the concentrations of trans-zeatin riboside throughout Arabidopsis plants and on the overall CK concentrations in roots, implying that AtENT3 participates in both the long- and the short-distance transport of CKs.

Ribosylated forms of plant hormones cytokinins (CKs) are the dominant CK species translocated at long distances. Their particular roles in plant physiology imply the existence of a yet uncharacterized CK riboside-specific membrane transport system. In this work, we report significant differences in the kinetics of the membrane transport of CK nucleobases and ribosides and the overall affinity of membrane-bound carriers towards the two CK forms. We show that CK ribosides can inhibit the uptake of CK nucleobases in tobacco Bright Yellow 2 cell suspensions but not vice versa, confirming the existence of a membrane transport system that strictly recognizes CK ribosides. We further characterize the membrane transport of CK nucleobases and ribosides mediated by AtENT3 (EQULIBRATIVE NUCLEOSIDE TRANSPORTER 3), showing its preference towards trans -zeatin riboside (tZR) over isopentenyl adenosine (iPR). With the molecular docking and molecular dynamics, we assess the interactions among the side chain of tZR and AtENT3 residues Tyr61 and Asp129, which are conserved in all AtENTs but not in the ENTs from non-plant species. Lastly, we show that atent3 mutation affects shoot phenotype, demonstrating the impact of CK riboside membrane transport on shoot development. ### Competing Interest Statement The authors have declared no competing interest. * ABC : ATP-BINDING CASSETTE ADFR : AutoDockFR software suite ARR : ARABIDOPSIS RESPONSE REGULATOR ANOVA : analysis of variance At : mouse-ear cress ( Arabidopsis thaliana ) AZG : AZA-GUANINE RESISTANT BA : benzyladenine BAR : benzyladenosine Bt : cattle ( Bos taurus ) BY-2 : Bright Yellow 2 CCCP : carbonyl cyanide 3-chlorophenylhydrazone CK : cytokinin CKX : CYTOKININ DEHYDROGENASE CMOS : complementary metal-oxide-semiconductor Col-0 Columbia-0 cZ : cis -zeatin DHZ : dihydrozeatin DHZR : dihydrozeatin riboside DiPy : dipyridamole DMSO : dimethyl sulfoxide Dr : zebrafish ( Danio rerio ) ENT : EQULIBRATIVE NUCLEOSIDE TRANSPORTER Hs : human ( Homo sapiens ) iP : isopentenyl adenine iPR : isopentenyl adenosine LOG : LONELY GUY MAD : median of absolute deviation Mm : mouse ( Mus musculus ) MS : Murashige-Skoog NBTI : S -(4-nitrobenzyl)-6-thioinosine NCBI : National Center for Biotechnology Information Os : rice ( Oryza sativa ) Pf : Plasmodium falciparum PUP : PURINE PERMEASE REL : relative expression level Rn : rat ( Ratus norvegicus ) SAM : shoot apical meristem Sc : yeast ( Saccharomyces cerevisiae ) SWEET : SUGAR WILL EVENTUALLY BE EXPORTED TRANSPORTER TM : transmembrane helix WUS : WUSCHEL tZ : trans -zeatin tZR : trans -zeatin riboside

To cope with biotic and abiotic stress conditions, land plants have evolved several levels of protection, including delicate defense mechanisms to respond to changes in the environment. The benefits of inducible defense responses can be further augmented by defense priming, which allows plants to respond to a mild stimulus faster and more robustly than plants in the naïve (non-primed) state. Priming provides a low-cost protection of agriculturally important plants in a relatively safe and effective manner. Many different organic and inorganic compounds have been successfully tested to induce resistance in plants. Among the plethora of commonly used physicochemical techniques, priming by plant growth regulators (phytohormones and their derivatives) appears to be a viable approach with a wide range of applications. While several classes of plant hormones have been exploited in agriculture with promising results, much less attention has been paid to cytokinin, a major plant hormone involved in many biological processes including the regulation of photosynthesis. Cytokinins have been long known to be involved in the regulation of chlorophyll metabolism, among other functions, and are responsible for delaying the onset of senescence. A comprehensive overview of the possible mechanisms of the cytokinin-primed defense or stress-related responses, especially those related to photosynthesis, should provide better insight into some of the less understood aspects of this important group of plant growth regulators.

Plasmodiophora brassicae is an obligate biotrophic pathogen causing clubroot disease in cruciferous plants. Infected plant organs are subject to profound morphological changes, the roots form characteristic galls, and the leaves are chlorotic and abscise. The process of gall formation is governed by timely changes in the levels of endogenous plant hormones that occur throughout the entire life cycle of the clubroot pathogen. The homeostasis of two plant hormones, cytokinin and auxin, appears to be crucial for club development. To investigate the role of cytokinin and auxin in gall formation, we used metabolomic and transcriptomic profiling of Arabidopsis thaliana infected with clubroot, focusing on the late stages of the disease, where symptoms were more pronounced. Loss-of-function mutants of three cytokinin receptors, AHK2, AHK3, and CRE1/AHK4, were employed to further study the homeostasis of cytokinin in response to disease progression; ahk double mutants developed characteristic symptoms of the disease, albeit with varying intensity. The most susceptible to clubroot disease was the ahk3 ahk4 double mutant, as revealed by measuring its photosynthetic performance. Quantification of phytohormone levels and pharmacological treatment with the cytokinin antagonist PI-55 showed significant changes in the levels of endogenous cytokinin and auxin, which was manifested by both enhanced and reduced development of disease symptoms in different genotypes.

Solubility of growth regulators is essential for their use in agriculture. Four new cytokinin salts─6-benzylaminopurine mesylate (1), 6-(2-hydroxybenzylamino)purine mesylate (2), 6-(3-hydroxybenzylamino)purine mesylate (3), and 6-(3-methoxybenzylamino)purine mesylate (4)─were synthesized, and their crystal structures were determined to clarify structural influence on water solubility. The mesylates were several orders of magnitude more water-soluble than the parent CKs. The new salts significantly reduced chlorophyll degradation and impairment of photosystem II functionality in barley leaf segments undergoing artificial senescence and had pronounced effects on the leaves' endogenous CK pools, maintaining high concentrations of functional metabolites for several days, unlike canonical CKs. A foliar treatment with 1 and 3 increased the harvest yield of spring barley by up to 8% when compared to treatment with the parent CKs while also increasing the number of productive tillers. This effect was attributed to the higher bioavailability of the mesylate salts and the avoidance of dimethyl sulfoxide exposure.

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

Cytokinins are plant hormones with biological functions ranging from coordination of plant growth to the regulation of biotic and abiotic stress-related responses and senescence. The components of the plant immune system can learn from past elicitations by microbial pathogens and herbivores and adapt to new threats. It is known that plants can enter the primed state of enhanced defense induced by either natural or synthetic compounds. While the involvement of cytokinins in defense priming has been documented, no comprehensive model of their action has been provided to date. Here, we report the functional characterization of two aromatic cytokinin derivatives, 6-benzylaminopurine-9-arabinosides (BAPAs), 3-methoxy-BAPA and 3-hydroxy-BAPA, that proved to be effective in delaying senescence in detached leaves while having low interactions with the cytokinin pathway. An RNA-seq profiling study on Arabidopsis leaves treated with 3-methoxy-BAPA revealed that short and extended treatments with this compound shifted the transcriptional response markedly toward defense. Both treatments revealed upregulation of genes involved in processes associated with plant innate immunity such as cell wall remodeling and upregulation of specific MAP kinases, most importantly MPK11, which is a MAPK module involved in stress-related signaling during the pathogen-associated molecular patterns (PAMPs) response. In addition, elevated levels of JA and its metabolites, jasmonate/ethylene-driven upregulation of PLANT DEFENSIN 1.2 (PDF1.2) and other defensins, and also temporarily elevated levels of reactive oxygen species marked the plant response to 3-methoxy-BAPA treatment. Synergistic interactions were observed when plants were cotreated with 3-hydroxy-BAPA and the flagellin-derived bacterial PAMP peptide (flg22), leading to the enhanced expression of the PAMP-triggered immunity (PTI) marker gene FRK1. Our data collectively show that some BAPAs can sensitively prime the PTI responses in a low micromolar range of concentrations while having no observable negative effects on the overall fitness of the plant.

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

Leaf senescence, accompanied by chlorophyll breakdown, chloroplast degradation and inhibition of photosynthesis, can be suppressed by an exogenous application of cytokinins. Two aromatic cytokinin arabinosides (6-benzylamino-9-β-d-arabinofuranosylpurines; BAPAs), 3-hydroxy- (3OHBAPA) and 3-methoxy- (3MeOBAPA) derivatives, have recently been found to possess high anti-senescence activity. Interestingly, their effect on the maintenance of chlorophyll content and maximal quantum yield of photosystem II (PSII) in detached dark-adapted leaves differed quantitatively in wheat (Triticum aestivum L. cv. Aranka) and Arabidopsis (Arabidopsisthaliana L. (Col-0)). In this work, we have found that the anti-senescence effects of 3OHBAPA and 3MeOBAPA in wheat and Arabidopsis also differ in other parameters, including the maintenance of carotenoid content and chloroplasts, rate of reduction of primary electron acceptor of PSII (QA) as well as electron transport behind QA, and partitioning of absorbed light energy in light-adapted leaves. In wheat, 3OHBAPA had a higher protective effect than 3MeOBAPA, whereas in Arabidopsis, 3MeOBAPA was the more efficient derivative. We have found that the different anti-senescent activity of 3OHBAPA and 3MeOBAPA was coupled to different ethylene production in the treated leaves: the lower the ethylene production, the higher the anti-senescence activity. 3OHBAPA and 3MeOBAPA also efficiently protected the senescing leaves of wheat and Arabidopsis against oxidative damage induced by both H2O2 and high-light treatment, which could also be connected with the low level of ethylene production.

The plant hormone cytokinin regulates various cell and developmental processes, including cell division and differentiation, embryogenesis, activity of shoot and root apical meristems, formation of shoot and root lateral organs and others 1 . Cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Based on the subcellular localization of cytokinin receptors in various transient expression systems, such as tobacco leaf epidermal cells, and membrane fractionation experiments of Arabidopsis and maize, the endoplasmic reticulum (ER) membrane has been proposed as a principal hormone perception site 2–4 . Intriguingly, recent study of the cytokinin transporter PUP14 has pointed out that the plasma membrane (PM)-mediated signalling might play an important role in establishment of cytokinin response gradients in various plant organs 5 . However, localization of cytokinin HK receptors to the PM, although initially suggested 6 , remains ambiguous. Here, by monitoring subcellular localizations of the fluorescently labelled natural cytokinin probe iP-NBD 7 and the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin fluoroprobes and receptors can enter the secretory pathway and reach the PM. We demonstrate that in cells of the root apical meristem, CRE1/AHK4 localizes to the PM and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. Our results provide a new perspective on cytokinin signalling and the possibility of multiple sites of perception at PM and ER, which may determine specific outputs of cytokinin signalling.

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.