Cytokinins (CKs), classically recognised as plant growth regulators, are increasingly implicated in plant-microbe interactions. Studies in herbaceous plants suggest that CKs can function as signalling molecules with dual roles in plant defence, either restricting or facilitating pathogen infection. However, their roles in tree-pathogen interactions remain largely unexplored. Here, we investigated CK induction, metabolism, and functions in black poplar (Populus nigra) during infection by the rust fungus Melampsora larici-populina, an obligate biotrophic pathogen. We found that most isoprenoid CKs accumulated rapidly in poplar leaves after rust inoculation but declined during the fungal proliferation and sporulation stages. In contrast, poplar-specific aromatic CKs, such as topolins, were induced throughout infection. Exogenous application of the predominant isoprenoid CK trans-zeatin (tZ) and poplar-specific aromatic CK (topolins) significantly enhanced rust colonisation. Elevated CKs reduced the accumulation of antifungal flavan-3-ols and defence-related hormones, including salicylic acid and jasmonates. Furthermore, increased CK levels, especially ortho-topolin, markedly increased free amino acid concentrations, potentially enhancing nutrient availability for the biotrophic rust. Collectively, our results reveal that poplar CKs weaken the tree's chemical defences while enhancing the accumulation of host-derived nutrients, thereby promoting infection by the rust fungus. These findings reveal a previously unexplored role of CKs in tree-pathogen interactions.

The excessive application of chemical fertilizers is a prevalent issue in modern agriculture, as it increases production costs, undermines economic returns, and degrades soil health. In this study, the effects of two compositionally distinct yeast derivatives, namely Yeast Glycoside Type I (YD1) and Water-Soluble Yeast Glycoside (YD2), on the growth, nutrient use efficiency, low-temperature (LT) stress tolerance, and rhizosphere microbial communities of pepper (Capsicum annuum L.) were investigated. The yeast derivatives (YD) were characterized using scanning electron microscopy (SEM) and biochemical analysis, which revealed distinct profiles: YD2 contained higher nitrogen (11.89%), isindole-3-acetic acid (IAA), and 1-Aminocyclopropane-1-carboxylic acid (ACC), whereas YD1 was enriched in cytokinins (isopentenyl adenine, iP; trans-zeatin, tZ). Under normal temperature (NT) conditions, application of YD2 at concentrations of 1% significantly enhanced pepper yield, shoot biomass, and nitrogen/potassium use efficiency, whereas YD1 exerted minimal effects. However, under low-temperature (LT) stress, applications of YD1 and YD2 did not significantly increase yield. Despite this, YD2 maintained root activity, mitigated chlorophyll degradation, and increased the activities of antioxidant enzymes under LT stress. Both YD altered rhizosphere bacterial community structure under NT conditions (PC1: 42.09%, p = 0.007); however, this effect was attenuated under LT stress (PC1: 24.85%, p = 0.003). Specifically, YD1 was associated with an increased relative abundance of Microscillaceae (NT) and Thiobacillus (LT), while YD2 was linked to an increase in Kaiserbacteria (NT) and Armatimonadales (LT). These findings suggest that the two YDs, particularly YD2, can influence pepper productivity and stress resilience through associations with improved nutrient uptake, physiological adjustments, and shifts in the rhizosphere microbiome. This pot-based study provides insights into the potential of tailored yeast derivatives as biostimulants, offering a basis for developing more sustainable agricultural practices.

Nitric oxide (NO) is a key signaling molecule regulating plant stress responses. In our study, the NO donor sodium nitroprusside (SNP) significantly promoted ginger seedling growth under salt stress, while treatment with the NO scavenger cPTIO resulted in an opposite response, supporting the involvement of NO in salt-stress responses. Transcriptome profiling of ginger leaves identified 2252 differentially expressed genes (DEGs), which were enriched in 55 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Further, analysis showed that 19 DEGs were enriched in plant hormone signal transduction, while 16 DEGs were enriched in starch and sugar metabolism. NO treatment increased the levels of indole-3-acetic acid (IAA), gibberellin 3 (GA3), and trans-zeatin (ZR), while reducing the levels of abscisic acid (ABA), methyl jasmonate (MeJA), and brassinosteroid (BR). Moreover, NO promoted the accumulation of soluble sugars and sucrose and enhanced the activities of key enzymes involved in starch and sucrose metabolism, thereby effectively improving osmotic adjustment. Collectively, NO improves antioxidant capacity, maintains endogenous phytohormone balance, and enhances osmotic adjustment, ultimately alleviating salt stress damage in ginger seedlings.

Purification of crude plant extracts by solid-phase extraction (SPE) is assumed to reduce matrix effect in phytohormone quantification by LC-MS/MS, but its efficacy is rarely assessed. We used postextraction spiking of isotope-labeled phytohormones to estimate matrix effect, recovery, and processes efficiency in extracts prepared according to protocols that implement SPE with tC18 and HLB sorbents, respectively, and according to a protocol that does not include SPE. Across six structurally diverse phytohormone classes and tomato fruit and leaf matrices, we did not find significant matrix effects associated with extracts prepared with no SPE. For recovery efficiency, process efficiency, and variation, we rarely found conclusive evidence that the methods that implemented SPE were optimal relative to the one that does not. A notable exception was fruit extracts prepared using HLB sorbents, which had minimal variation for indole-3-acetic acid, trans-zeatin, and trans-zeatin riboside, all of which were detected with positive electrospray ionization.

Salt stress is frequently encountered by plants and is a major constraint on crop production. Wheat (Triticum aestivum L.) is a staple cereal crop that is important for human health and food security. However, the response mechanisms of roots from different wheat varieties to salt stress remain unclear. In this study, we analyzed the complex response mechanisms of the salt-tolerant variety Longjian 114 (LJ114) and the salt-sensitive variety Chinese Spring (CS) to salt stress using integrated transcriptomic and metabolomic analyses. The results showed that LJ114 maintained better growth and exhibited higher antioxidant enzyme activities and greater osmotic adjustment capacity under salt stress. Differentially expressed genes in LJ114 were predominantly enriched in flavonoid biosynthesis, alpha-linolenic acid metabolism, and zeatin biosynthesis under salt stress. Metabolomic profiling identified 1366 differential metabolites, with LJ114 specifically enriched in tyrosine metabolism, tryptophan metabolism, glycerophospholipid metabolism, and fatty acid metabolism under salt stress. Integrated analyses further highlighted ABC transporters and alanine, aspartate, and glutamate metabolism as hub pathways, with notably higher GAD expression and GABA accumulation under salt stress. These findings suggest new insights into the molecular mechanisms underlying wheat salt tolerance and propose potential candidate targets for enhancing crop salinity tolerance.

Postmenopausal osteoporosis (PMOP) is a metabolic bone disorder driven by excessive osteoclast (OC) activity, but current therapies are limited by adverse effects. Trans-zeatin (TZ), a naturally occurring plant cytokinin with antioxidant properties, has not been investigated in bone metabolism. Here, we show that TZ suppresses receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption in vitro and attenuates ovariectomy-induced trabecular bone loss in mice. Mechanistically, TZ activates nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant responses by upregulating heme oxygenase-1 (HO-1) and catalase while suppressing NADPH oxidase 4 (NOX4) expression, thereby reducing reactive oxygen species (ROS) accumulation. This redox modulation subsequently dampens protein kinase B (AKT) signaling, downregulates the c-Fos/nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) transcriptional program, and decreases the expression of OC-specific effector genes. Notably, Nrf2 knockdown partially reverses the inhibitory effects of TZ, whereas concurrent AKT knockdown attenuates this rescue effect, suggesting that AKT is an important downstream signaling component associated with Nrf2-mediated redox regulation. Collectively, these findings identify TZ as a promising natural candidate for PMOP intervention through coordinated regulation of redox homeostasis and AKT-dependent osteoclastogenic signaling.

Mulch drip fertigation strategies have been widely adopted to improve maize productivity in semi-arid regions. However, the impact of the mulch-based drip fertigation on endogenous hormone dynamics in crops and grain yield has not been fully elucidated, and the biochemical mechanisms underlying yield improvement under different drip fertigation strategies remain unclear. Grain filling has a significant impact on maize production. The aim of this study was to explore the effect of plastic film mulching materials on maize grain filling under a drip fertigation strategy, and the relationship between this effect and changes in endogenous hormones. This study compared biodegradable film mulching (BM) with soil crust ridges (SR) under high (H), medium (M), and low (L) drip irrigation conditions, and measured that the grain filling characteristics and changes in grain endogenous hormones in various parts of maize ears during the growth stage. The results showed that the grain filling rate, dry matter plant[-1], hormone changes, yield components, water use efficiency (WUE), and ET of maize under the BMH treatment were significantly higher than those under the SR treatment. BMH treatment significantly increased soil water storage significantly increased plant hormone contents such as abscisic acid (ABA), indole-3-Acetic acid (IAA), and zeatin + Zeatin Riboside (Z+ZR), and decreased the evolution rate of gibberellins (Gas) and ethylene, thereby increasing maize yield. These results indicate that BMH treatment improved the filling process, soil respiration rate, WUE, yield, and yield composition of maize. High drip fertigation with BM or SR treatment significantly affected endogenous hormone changes, thereby regulating the activity grain-filling period (AGP), maximum grain-filling rate (Gmax), maximum grain weight (Wmax), mean grain-filling rates (Gmean) and occurrence time of maximal filling rate (Tmax) of maize. In addition, high drip fertigation and the BM strategy significantly increased soil water storage and soil respiration rate during the grain filling period. Based on these results, we conclude that BMH significantly improved soil water storage, thereby regulating the maize grain filling rate, which was significantly correlated with the balance of endogenous hormones in the grains.

An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.

Ruscus hypoglossum L. is an evergreen ornamental species valued as cut foliage, but its commercial production is constrained by slow vegetative propagation and limited availability of uniform planting material. This study aimed to develop an efficient in vitro propagation protocol for R. hypoglossum by evaluating the effects of different cytokinins and spermidine on shoot organogenesis and subsequent rooting. Thidiazuron (TDZ), zeatin, kinetin, and spermidine were applied at 0.5 and 1.0 mg L[-1] to rhizome explants derived from in vitro-grown seedlings. Explant length, shoot and cladode number, fresh weight, shoot diameter, chlorophyll a and b, total carotenoid content, SPAD value, and rooting performance were assessed. Significant treatment effects were observed for most traits. Kinetin at 1.0 mg L[-1] produced the highest shoot proliferation (4.66 shoots explant[-1]), cladode number (12.00 explant[-1]), fresh weight (0.31 g), chlorophyll a (0.72 mg g[-1] FW), chlorophyll b (0.29 mg g[-1] FW), and SPAD value (40.25). Spermidine at 1.0 mg L[-1] resulted in the highest carotenoid content (0.59 mg g[-1] FW) and promoted greater explant elongation, whereas TDZ at 1.0 mg L[-1] reduced cladode formation and pigment accumulation. Multivariate analyses consistently identified kinetin as the most favorable treatment during multiplication. Shoots regenerated with 1.0 mg L[-1] kinetin also showed the highest rooting response following a 10 s pulse treatment with 2.0 mg L[-1] IBA and transfer to hormone-free half-strength MS medium. Overall, kinetin was the most effective treatment under the conditions tested, providing a practical basis for efficient clonal propagation and ex situ conservation of R. hypoglossum.

Cytokinins (CKs) are a class of phytohormones that regulate a wide range of developmental processes in plants. Different structural forms of these phytohormones have been widely used in plant tissue culture, although comparative studies on their temporal effects on gene expression remain limited. In this study, we compared the effects of trans-zeatin (tZ), meta-topolin (mT), and thidiazuron (TDZ) on the expression dynamics of ten CK-responsive genes associated with shoot growth and development in Arabidopsis thaliana. Ten-day-old Arabidopsis seedlings were exposed to liquid nutrient media containing 5 μM tZ, mT, or TDZ, or a control medium, for 15 min and 1, 2, 4, 8, and 24 h. Gene expression was analyzed by RT-qPCR using three independent biological replicates. All tested genes were strongly upregulated at 4 h following mT treatment, whereas most genes showed the highest expression at 8 h in response to TDZ. In contrast, tZ generally induced weaker and slower transcriptional responses. Expression levels of all analyzed genes returned to control-like or lower levels within 24 h. Overall, the three structurally distinct cytokinins induced markedly different temporal transcriptional responses, with mT producing the fastest and most pronounced effects on the expression of the tested genes under the applied experimental conditions. These findings improve our understanding of CK-dependent transcriptional dynamics during shoot development and provide a basis for future studies on CK-regulated morphogenesis and plant regeneration.

Clubroot, caused by Plasmodiophora brassicae, poses a serious threat to the rapeseed (Brassica napus) industry. Due to B. napus being an allopolyploid with a complex genome and the current scarcity of available resistance gene resources, the molecular basis of rapeseed resistance to P. brassicae remains poorly understood. Here, we performed a functional characterization of BnEIN2 (ethylene-insensitive protein) to explore the role of ethylene signaling in rapeseed resistance to P. brassicae. The Bnein2 mutants generated through CRISPR/Cas9 technology exhibited enhanced resistance to P. brassicae, along with reduced 1-aminocyclopropane-1-carboxylic acid (ACC)/S-adenosyl-L-methionine (SAM) accumulation and ethylene insensitivity. Pharmacological assays demonstrated that inhibitors of ethylene biosynthesis or signaling improved the resistance of Bnein2 mutant plants to P. brassicae. Transcriptome analysis revealed that loss-of-function of BnEIN2 affected the expression of ethylene-, auxin-, and cytokinin-related genes. Moreover, the increased resistance of Bnein2 mutants to P. brassicae was accompanied by a reduction in auxin (indole-3-acetic acid, IAA) biosynthesis and degradation of cytokinin (trans-zeatin, TZ). Collectively, these findings establish the negative regulatory role of ethylene signaling in rapeseed resistance to P. brassicae. This study represents the first effort to elucidate rapeseed resistance to P. brassicae by directly obtaining rapeseed genetic material and offer novel insights into the hormonal regulatory network underlying disease resistance and valuable resources for breeding clubroot-resistant varieties.

5-Aminolevulinic acid (ALA) is an emerging plant growth regulator, yet its mechanisms in fruit development remain unclear. Here, we show that ALA promotes early growth and ultimate size in peach (Prunus persica) fruits. ALA treatment significantly enhanced the mesocarp area and the cell number, accompanied by elevated levels of indole-3-acetic acid (IAA) and trans-zeatin riboside. Transcriptomic analysis identified PpARF9 as a key transcriptional factor, regulating auxin homeostasis and responsive to ALA treatment. Functional studies with yeast-one-hybrid (Y1H), dual-luciferase (DLR), electrophoretic mobility shift assay (EMSA) and homogenous genetic transformation revealed that PpARF9 directly transcriptionally activates PpGH3.1, a crucial gene which encodes chloroplastic auxin amide synthetase to conjugate free IAA and repress cell division. Overexpression of PpGH3.1 reduced free IAA with more IAA conjugate accumulation, thereby suppressing cell cycle. Conversely, silencing PpGH3.1 with RNA interference had opposite effects. Notably, exogenous ALA alleviated the inhibitory effects of PpARF9 and PpGH3.1 on cell proliferation. These findings indicate that ALA promotes cell division by downregulating the PpARF9-PpGH3.1 module to maintain higher levels of active IAA, facilitating cell division, increasing cell number and fruit size. These provide a mechanistic insight into ALA-regulated fruit development and support its application in improving fruit production.