Phytochrome-dependent responsiveness to root-derived cytokinins enables coordinated elongation responses to combined light and nitrate cues.

Plants growing at high densities can detect competitors through changes in the composition of light reflected by neighbours. In response to this far-red-enriched light, plants elicit adaptive shade avoidance responses for light capture, but these need to be balanced against other input signals, such as nutrient availability. Here, we investigated how Arabidopsis integrates shade and nitrate signalling. We unveiled that nitrate modulates shade avoidance via a previously unknown shade response pathway that involves root-derived trans-zeatin (tZ) signal and the BEE1 transcription factor as an integrator of light and cytokinin signalling. Under nitrate-sufficient conditions, tZ promotes hypocotyl elongation specifically in the presence of supplemental far-red light. This occurs via PIF transcription factors-dependent inhibition of type-A ARRs cytokinin response inhibitors. Our data thus reveal how plants co-regulate responses to shade cues with root-derived information about nutrient availability, and how they restrict responses to this information to specific light conditions in the shoot.

Crosstalk among plant hormone regulates the root development.

The plant root absorbs water and nutrients, anchors the plant in the soil, and promotes plant development. Root is developed from root apical meristem (RAM), which is formed during embryo stage and is maintained by dividing stem cells. Plant hormones have a predominant role in RAM maintenance. This review evaluates the functional crosstalk among three major hormones (auxin, cytokinin, and brassinolide) in RAM development in Arabidopsis, integrating a variety of experimental data into a regulatory network and revealing multiple layers of complexity in the crosstalk among these three hormones. We also discuss possible directions for future research on the roles of hormones in regulating RAM development and maintenance.

Nutrient competition between female cones and young seeds in spring affects the physiological dropping and nut-setting rate in Torreya grandis.

Physiological seed drop is a recognized phenomenon in economic forest, caused by the abscission of developing seeds due to intergroup competition for resources. However, little is known about the resource allocation dynamics in species exhibiting a biennial fruiting cycle, where interactions occur not only among seeds of the same year but also between reproductive structures from consecutive years. In this study, we explored the dynamics of resource allocation in Torreya grandis, a nut crop with a prototypical two-year seed development pattern. We implemented thinning treatments of 0%, 30%, and 60% on female cones and/or immature seeds during the spring, targeting various stages of development both pre- and post-pollination. Our findings reveal a pronounced resource competition in Torreya, evidenced by a natural seed-setting rate of merely 9.4%. Contrary to expectations, seed thinning did not lead to an obvious increase in nut-setting rates, whereas a substantial increase to 20.5% was observed when female cones were thinned by 60% at 20 days before pollination. The cone thinning treatment appears to have influenced seed development through positive cytokinin and negative abscisic acid effects. This indicates that intergroup competition between female cones and nuts is a more significant factor in seed drop than inner nut competition, and there seems to be an interaction between the two groups. We demonstrate that, in Torreya with biennial seed development, it is the competition between female cones and immature seeds that is important. This insight expands our comprehension of the physiological mechanisms governing seed drop in biennial fruiting species and managing the reproductive organ load to optimize nutrient allocation.

Cytokinin catabolism and transport are involved in strigolactone-modulated rice tiller bud elongation fueled by phosphate and nitrogen supply.

Phosphate (P) and nitrogen (N) fertilization affect rice tillering, indicating that P- and N-regulated tiller growth has a crucial effect on grain yield. Cytokinins and strigolactones (SLs) promote and inhibit tiller bud outgrowth, respectively; however, the underlying mechanisms are unclear. In this study, tiller bud outgrowth and cytokinin fractions were evaluated in rice plants fertilized at different levels of P and N. Low phosphate or nitrogen (LP or LN) reduced rice tiller numbers and bud elongation, in line with low cytokinin levels in tiller buds and xylem sap as well as low TCSn:GUS expression, a sensitive cytokinin signal reporter, in the stem base. Furthermore, exogenous cytokinin (6-benzylaminopurin, 6-BA) administration restored bud length and TCSn:GUS activity in LP- and LN-treated plants to similar levels as control plants. The TCSn:GUS activity and tiller bud outgrowth were less affected by LP and LN supplies in SL-synthetic and SL-signaling mutants (d17 and d53) compared to LP- and LN-treated wild-type (WT) plants, indicating that SL modulate tiller bud elongation under LP and LN supplies by reducing the cytokinin levels in tiller buds. OsCKX9 (a cytokinin catabolism gene) transcription in buds and roots was induced by LP, LN supplies and by adding the SL analog GR24. A reduced response of cytokinin fractions to LP and LN supplies was observed in tiller buds and xylem sap of the d53 mutant compared to WT plants. These results suggest that cytokinin catabolism and transport are involved in SL-modulated rice tillering fueled by P and N fertilization.

Functional characterization of WsPR-1 reveals its interplay with cytokinin and gibberellin signaling pathways.

Pathogenesis-related protein 1 (PR-1) is an antimicrobial protein involved in systemic acquired resistance (SAR) in plants, but its regulatory role and interactions with other pathways remain unclear. In this study, we functionally characterize WsPR-1 gene of Withania somnifera in Nicotiana tabacum to elucidate its role in plant defense, growth, and development. Interestingly, transgenic tobacco plants with increased levels of cytokinin (CK) and decreased gibberellins (GAs) exhibited stunted shoot growth, an underdeveloped root system, modified leaf morphology, reduced seed pod production, and delayed leaf senescence. Transcriptional analysis revealed that WsPR-1 overexpression downregulated the GA 20-oxidase (GA20ox) gene involved in GA biosynthesis while upregulating GA 2-oxidase (GA2ox), a GA catabolic enzyme. Moreover, transcript levels of FRUITFULL (FUL) and LEAFY (NFL2) flowering genes exhibited a decrease in WsPR-1 plants, which could explain the delayed flowering and reduced seed pod development in transgenic plants. Confocal microscopy confirmed increased lignin deposition in stem cross-sections of WsPR-1 transgenic plants, supported by gene expression analysis and lignin content quantification. Additionally, our findings also suggest the involvement of Knotted1-like homeobox (KNOX) gene in enhancing cytokinin levels. This study highlights PR-1's regulatory role in plant growth and development, with potential to boost crop yields and enhance resilience.

At the crossroads: strigolactones mediate changes in cytokinin synthesis and signalling in response to nitrogen limitation.

Strigolactones (SLs) are key regulators of shoot growth and responses to environmental stimuli. Numerous studies have indicated that nitrogen (N) limitation induces SL biosynthesis, suggesting that SLs may play a pivotal role in coordinating systemic responses to N availability, but this idea has not been clearly demonstrated. Here, we generated triple knockout mutants in the SL synthesis gene TaDWARF17 (TaD17) in bread wheat and investigated their phenotypic and transcriptional responses under N limitation, aiming to elucidate the role of SLs in the adaptation to N limitation. Tad17 mutants display typical SL mutant phenotypes, and fail to adapt their shoot growth appropriately to N. Despite exhibiting an increased tillering phenotype, Tad17 mutants continued to respond to N limitation by reducing tiller number, suggesting that SLs are not the sole regulators of tillering in response to N availability. RNA-seq analysis of basal nodes revealed that the loss of D17 significantly altered the transcriptional response of N-responsive genes, including changes in the expression profiles of key N response master regulators. Crucially, our findings suggest that SLs are required for the transcriptional downregulation of cytokinin (CK) synthesis and signalling in response to N limitation. Collectively, our results suggest that SLs are essential for the appropriate morphological and transcriptional adaptation to N limitation in wheat, and that the repressive effect of SLs on shoot growth is partly mediated by their repression of CK synthesis.

The TaWAK2-TaNAL1-TaDST pathway regulates leaf width via cytokinin signaling in wheat.

Leaves play a crucial role in photosynthesis and respiration, ultimately affecting the final grain yield of crops, including wheat (Triticum aestivum L.); however, the molecular mechanisms underlying wheat leaf development remain largely unknown. Here, we isolated a narrow-leaf gene, TaWAK2-A, through a map-based cloning strategy. TaWAK2-A encodes a wall-associated kinase (WAK), for which a single Ala-to-Val amino acid substitution reduces the protein stability, leading to a narrow-leaf phenotype in wheat. Further investigation suggests that TaWAK2 directly interacts with and phosphorylates TaNAL1, a trypsin-like serine/cysteine protease. The phosphorylated TaNAL1 is then involved in the degradation of the zinc finger transcription factor TaDST, which acts as a repressor of leaf expansion by activating the expression of the cytokinin oxidase gene TaCKX9 and triggering in vivo cytokinin degradation. Therefore, our findings elucidate a signaling cascade involving TaWAK2-TaNAL1-TaDST that sheds light on the regulation of wheat leaf development.

Cytokinin and ALOG proteins regulate pluripotent stem cell identity in the moss Physcomitrium patens.

The shoot apical meristem (SAM) contains pluripotent stem cells that produce all the aerial parts of the plant. Stem cells undergo asymmetric cell divisions to self-renew and to produce differentiating cells. Our research focused on unraveling the mechanisms governing the specification of these two distinct cell fates following the stem cell division. For this purpose, we used the model organism Physcomitrium patens, which features a singular pluripotent stem cell known as the gametophore apical cell. We show that the activity of cytokinins, critical stem cell regulators, is restricted to the gametophore apical cell due to the specific localization of PpLOG, the enzyme responsible for cytokinin activation. In turn, PpTAW, which promotes differentiating cell identity of the merophyte, is excluded from the gametophore apical cell by the action of cytokinins. We propose a cytokinin-based model for the establishment of asymmetry in the pluripotent stem cell division.

Search for Expression Marker Genes That Reflect the Physiological Conditions of Blossom End Enlargement Occurrence in Cucumber.

Blossom end enlargement (BEE) is a postharvest deformation that may be related to the influx of photosynthetic assimilates before harvest. To elucidate the mechanism by which BEE occurs, expression marker genes that indicate the physiological condition of BEE-symptomatic fruit are necessary. First, we discovered that preharvest treatment with a synthetic cytokinin, N-(2-Chloro-4-pyridyl)-N'-phenylurea (CPPU), promoted fruit growth and suppressed BEE occurrence. This suggests that excessive assimilate influx is not a main cause of BEE occurrence. Subsequently, the expression levels of seven sugar-starvation marker genes, CsSEF1, AS, CsFDI1, CsPID, CsFUL1, CsETR1, and CsERF1B, were compared among symptomatic and asymptomatic fruits, combined with and without CPPU treatment. Only CsSEF1 showed a higher expression level in asymptomatic fruits than in symptomatic fruits, regardless of CPPU treatment. This was then tested using fruits stored via the modified-atmosphere packaging technique, which resulted in a lower occurrence of BEE, and the asymptomatic fruits showed a higher CsSEF1 expression level than symptomatic fruits, regardless of the packaging method. CsSEF1 codes a CCCH-type zinc finger protein, and an increase in the expression of CsSEF1 was correlated with a decrease in the fruit respiration rate. Thus, CsSEF1 may be usable as a BEE expression marker gene.

Spider mite infestation triggers coordinated hormonal trade-offs enabling plant survival with a fitness cost.

Tetranychus urticae is an important pest that causes severe damage to a wide variety of plants and crops, leading to a substantial productivity loss. Previous research has been focused on plant defence response to T. urticae to improve plant resistance. However, plant growth, development and reproduction throughout the infestation process have not been previously studied. Through physiological, biochemical, transcriptomic and hormonomic evaluation, we uncover the molecular mechanisms directing the defence-growth trade-off established in Arabidopsis upon T. urticae infestation. Upon mite attack, plants suffer an adaptation process characterized by a temporal separation between the defence and growth responses. Jasmonic and salicylic acids regulate the main defence responses in combination with auxin and abscisic acid. However, while the reduction of both auxin signalling and gibberellin, cytokinin and brassinosteroid biosynthesis lead to initial growth arrest, increasing levels of growth hormones at later stages enables growth restart. These alterations lead to a plant developmental delay that impacts both seed production and longevity. We demonstrate that coordinated trade-offs determine plant adaptation and survival, revealing mite infestation has a long-lasting effect negatively impacting seed viability. This study provides additional tools to design pest management strategies that improve resistance without penalty in plant fitness.

Adaxial-abaxial bipolar leaf genes encode a putative cytokinin receptor and HD-Zip III, and control the formation of ectopic shoot meristems in rice.

Shoot apical meristems (SAMs) continuously initiate organ formation and maintain pluripotency through dynamic genetic regulations and cell-to-cell communications. The activity of meristems directly affects the plant's structure by determining the number and arrangement of organs and tissues. We have taken a forward genetic approach to dissect the genetic pathway that controls cell differentiation around the SAM. The rice mutants, adaxial-abaxial bipolar leaf 1 and 2 (abl1 and abl2), produce an ectopic leaf that is fused back-to-back with the fourth leaf, the first leaf produced after embryogenesis. The abaxial-abaxial fusion is associated with the formation of an ectopic shoot meristem at the adaxial base of the fourth leaf primordium. We cloned the ABL1 and ABL2 genes of rice by mapping their chromosomal positions. ABL1 encodes OsHK6, a histidine kinase, and ABL2 encodes a transcription factor, OSHB3 (Class III homeodomain leucine zipper). Expression analyses of these mutant genes as well as OSH1, a rice ortholog of the Arabidopsis STM gene, unveiled a regulatory circuit that controls the formation of an ectopic meristem near the SAM at germination.

Cytokinin oxidase2-deficient mutants improve panicle and grain architecture through cytokinin accumulation and enhance drought tolerance in indica rice.

KEY MESSAGE: The Osckx2 mutant accumulates cytokinin thereby enhancing panicle branching, grain yield, and drought tolerance, marked by improved survival rate, membrane integrity, and photosynthetic function. Cytokinins (CKs) are multifaceted hormones that regulate growth, development, and stress responses in plants. Cytokinins have been implicated in improved panicle architecture and grain yield; however, they are inactivated by the enzyme cytokinin oxidase (CKX). In this study, we developed a cytokinin oxidase 2 (Osckx2)-deficient mutant using CRISPR/Cas9 gene editing in indica rice and assessed its function under water-deficit and salinity conditions. Loss of OsCKX2 function increased grain number, secondary panicle branching, and overall grain yield through improved cytokinin content in the panicle tissue. Under drought conditions, the Osckx2 mutant conserved more water and demonstrated improved water-saving traits. Through reduced transpiration, Osckx2 mutants showed an improved survival response than the wild type to unset dehydration stress. Further, Osckx2 maintained chloroplast and membrane integrity and showed significantly improved photosynthetic function under drought conditions through enhanced antioxidant protection systems. The OsCKX2 function negatively affects panicle grain number and drought tolerance, with no discernible impact in response to salinity. The finding suggests the utility of the beneficial Osckx2 allele in breeding to develop climate-resilient, high-yielding cultivars for future food security.

Adaptive roles of cytokinins in enhancing plant resilience and yield against environmental stressors.

Innovative agricultural strategies are essential for addressing the urgent challenge of food security in light of climate change, population growth, and various environmental stressors. Cytokinins (CKs) play a pivotal role in enhancing plant resilience and productivity. These compounds, which include isoprenoid and aromatic types, are synthesized through pathways involving key enzymes such as isopentenyl transferase and cytokinin oxidase. Under abiotic stress conditions, CKs regulate critical physiological processes by improving photosynthetic efficiency, enhancing antioxidant enzyme activity, and optimizing root architecture. They also reduce the levels of reactive oxygen species and malondialdehyde, resulting in improved plant performance and yield. CKs interact intricately with other phytohormones, including abscisic acid, ethylene, salicylic acid, and jasmonic acid, to modulate stress-responsive pathways. This hormonal cross-talk is vital for finely tuning plant responses to stress. Additionally, CKs influence nutrient uptake and enhance responses to heavy metal stress, thereby bolstering overall plant resilience. The application of CKs helps plants maintain higher chlorophyll levels, boost antioxidant systems, and promote root and shoot growth. The strategic utilization of CKs presents an adaptive approach for developing robust crops capable of withstanding diverse environmental stressors, thus contributing to sustainable agricultural practices and global food security. Ongoing research into the mechanisms of CK action and their interactions with other hormones is essential for maximizing their agricultural potential. This underscores the necessity for continued innovation and research in agricultural practices, in alignment with global goals of sustainable productivity and food security.

OsNAC121 regulates root development, tillering, panicle morphology, and grain filling in rice plant.

Transcription factors in coordination with phytohormones form an intricate regulatory network modulating vital cellular mechanisms like development, growth and senescence in plants. In this study, we have functionally characterized the transcription factor OsNAC121 by developing gene silencing and overexpressing transgenic rice plants, followed by detailed analyses of the plant architecture. Transgenic lines exhibited remodelling in crown root development, lateral root structure and density, tiller height and number, panicle and grain morphologies, underpinning the imbalanced auxin: cytokinin ratio due to perturbed auxin transportation. Application of cytokinin, auxin and abscisic acid increased OsNAC121 gene expression nearly 17-, 6- and 91-folds, respectively. qRT-PCR results showed differential expressions of auxin and cytokinin pathway genes, implying their altered levels. A 47-fold higher expression level of OsNAC121 during milky stage in untransformed rice, compared to 14-day old shoot tissue, suggests its crucial role in grain filling; as evidenced by a large number of undeveloped grains produced by the gene silenced lines. Crippled gravitropic response by the transgenic plants indicates their impaired auxin transport. Bioinformatics revealed that OsNAC121 interacts with co-repressor (TOPLESS) proteins and forms a part of the inhibitor complex OsIAA10, an essential core component of auxin signalling pathway. Therefore, OsNAC121 emerges as an important regulator of various aspects of plant architecture through modulation of crosstalk between auxin and cytokinin, altering their concentration gradient in the meristematic zones, and consequently modifying different plant organogenesis processes.

PagKNAT2/6b regulates tension wood formation and gravitropism by targeting cytokinin metabolism.

Tension wood is a specialized xylem tissue associated with gravitropism in angiosperm trees. However, few regulators of tension wood formation have been identified. The molecular mechanisms underpinning tension wood formation remain elusive. Here, we report that a Populus KNOTTED-like homeobox gene, PagKNAT2/6b, is involved in tension wood formation and gravity response. Transgenic poplar plants overexpressing PagKNAT2/6b displayed more sensitive gravitropism than controls, as indicated by increased stem curvature. Microscopic examination revealed greater abundance of fibre cells with a gelatinous cell wall layer (G-layer) and asymmetric growth of secondary xylem in PagKNAT2/6b overexpression lines. Conversely, PagKNAT2/6b dominant repression plants exhibited decreased tension wood formation and reduced response to gravity stimulation. Moreover, sensitivity to gravity stimulation showed a negative relationship with development stage. Expression of genes related to growth and senescence was affected in PagKNAT2/6b transgenic plants. More importantly, transcription activation and electrophoretic mobility shift assays suggested that PagKNAT2/6b promotes the expression of cytokinin metabolism genes. Consistently, cytokinin content was increased in PagKNAT2/6b overexpression plants. Therefore, PagKNAT2/6b is involved in gravitropism and tension wood formation, likely via modulation of cytokinin metabolism.

Cytokinin and reproductive shoot architecture: bigger and better?

Cytokinin (CK) is a key plant hormone, but one whose effects are often misunderstood, partly due to reliance on older data from before the molecular genetic age of plant science. In this mini-review, we examine the role of CK in controlling the reproductive shoot architecture of flowering plants. We begin with a long overdue re-examination of the role of CK in shoot branching, and discuss the relatively paucity of genetic evidence that CK does play a major role in this process. We then examine the role of CK in determining the number of inflorescences, flowers, fruit and seed that plants initiate during reproductive development, and how these are arranged in space and time. The genetic evidence for a major role of CK in controlling these processes is much clearer, and CK has profound effects in boosting the size and number of most reproductive structures. Conversely, the attenuation of CK levels during the reproductive phase likely contributes to reduced organ size seen later in flowering, and the ultimate arrest of inflorescence meristems during end-of-flowering. We finish by discussing how this information can potentially be used to improve crop yields.

AZGs: a new family of cytokinin transporters.

Cytokinins (CKs) are phytohormones structurally similar to purines that play important roles in various aspects of plant physiology and development. The local and long-distance distribution of CKs is very important to control their action throughout the plant body. Over the past decade, several novel CK transporters have been described, many of which have been linked to a physiological function rather than simply their ability to transport the hormone in vitro. Purine permeases, equilibrative nucleotide transporters and ATP-binding cassette transporters are involved in the local and long-range distribution of CK. In addition, members of the Arabidopsis AZA-GUANINE RESISTANT (AZG) protein family, AZG1 and AZG2, have recently been shown to mediate CK uptake at the plasma membrane and endoplasmic reticulum. Despite sharing ∼50% homology, AZG1 and AZG2 have unique transport mechanisms, tissue-specific expression patterns, and subcellular localizations that underlie their distinct physiological functions. AZG2 is expressed in a small group of cells in the overlying tissue around the lateral root primordia, where its expression is induced by auxins and it is involved in the regulation of lateral root growth. AZG1 is ubiquitously expressed, with high levels in the division zone of the root apical meristem. Here, it binds and stabilises the auxin efflux carrier PIN1, thereby shaping root architecture, particularly under salt stress. This review highlights the latest findings on the protein properties, transport mechanisms and cellular functions of this new family of CK transporters and discusses perspectives for future research in this field.

VlMYB4 and VlCDF3 co-targeted the VlLOG11 promoter to regulate fruit setting in grape (Vitis vinifera L).

KEY MESSAGE: The VlLOG11 mediates the cytokinin signaling pathway to regulate grape fruit setting. Fruit set, as an accepted agronomic trait, is inextricably linked with fruit quality and yield. Previous studies have demonstrated that exogenous treatment with the synthetic cytokinin analog, forchlorfenuron (CPPU), significantly enhances fruit set. In this study, a significant reduction in endogenous cytokinins was found by measuring the content of cytokinins in young grape berries after CPPU treatment. LONELY GUYs (VlLOGs), a key cytokinin-activating enzyme working in the biosynthesis pathway of cytokinins, exhibited differential expression. Some differentially expressed VlLOGs genes were presented by RNA seq data and their functions and regulation patterns were further investigated. The results showed that VlLOG11 was differentially expressed in young grape berries after CPPU treatment. Overexpression of VlLOG11 in tomato increases the amount of fruit set, and upregulated the expression of genes associated with cytokinin signaling including SlHK4, SlHK5, SlHP3, SlHP4, SlPHP1, SlPHP2. VlMYB4 and VlCDF3 could regulate the expression of VlLOG11 by directly binding to its promoter in young grape berries during fruit set. These results strongly demonstrated that VlMYB4/VlCDF3-VlLOG11 regulatory module plays a key role in the process of fruit setting in grape. This provided a basis for the molecular mechanism of VlLOG11-mediated cytokinin biosynthesis in young grape fruit set.

Genome-Wide Investigation of the CRF Gene Family in Maize and Functional Analysis of ZmCRF9 in Response to Multiple Abiotic Stresses.

The cytokinin response factors (CRFs) are pivotal players in regulating plant growth, development, and responses to diverse stresses. Despite their significance, comprehensive information on CRF genes in the primary food crop, maize, remains scarce. In this study, a genome-wide analysis of CRF genes in maize was conducted, resulting in the identification of 12 members. Subsequently, we assessed the chromosomal locations, gene duplication events, evolutionary relationships, conserved motifs, and gene structures of all ZmCRF members. Analysis of ZmCRF promoter regions indicated the presence of cis-regulatory elements associated with plant growth regulation, hormone response, and various abiotic stress responses. The expression patterns of maize CRF genes, presented in heatmaps, exhibited distinctive patterns of tissue specificity and responsiveness to multiple abiotic stresses. qRT-PCR experiments were conducted on six selected genes and confirmed the involvement of ZmCRF genes in the plant's adaptive responses to diverse environmental challenges. In addition, ZmCRF9 was demonstrated to positively regulate cold and salt tolerance. Ultimately, we explored the putative interaction partners of ZmCRF proteins. In summary, this systematic overview and deep investigation of ZmCRF9 provides a solid foundation for further exploration into how these genes contribute to the complex interplay of plant growth, development, and responses to stress.

Integrated Methylome and Transcriptome Analysis between Wizened and Normal Flower Buds in Pyrus pyrifolia Cultivar 'Sucui 1'.

Here, cytosine methylation in the whole genome of pear flower buds was mapped at a single-base resolution. There was 19.4% methylation across all sequenced C sites in the Pyrus pyrifolia cultivar 'Sucui 1' flower bud genome. Meantime, the CG, CHG, and CHH sequence contexts (where H = A, T or C) exhibited 47.4%, 33.3%, and 11.9% methylation, respectively. Methylation in different gene regions was revealed through combining methylome and transcriptome analysis, which presented various transcription trends. Genes with methylated promoters exhibited lower expression levels than genes with non-methylated promoters, while body-methylated genes displayed an obvious negative correlation with their transcription levels. The methylation profiles of auxin- and cytokinin-related genes were estimated. And some of them proved to be hypomethylated, with increased transcription levels, in wizened buds. More specifically, the expression of the genes PRXP73, CYP749A22, and CYP82A3 was upregulated as a result of methylation changes in their promoters. Finally, auxin and cytokinin concentrations were higher in wizened flower buds than in normal buds. The exogenous application of paclobutrazol (PP333) in the field influenced the DNA methylation status of some genes and changed their expression level, reducing the proportion of wizened flower buds in a concentration-dependent manner. Overall, our results demonstrated the relationship between DNA methylation and gene expression in wizened flower buds of P. pyrifolia cultivar 'Sucui 1', which was associated with changes in auxin and cytokinin concentrations.