Hormesis management of Moringa oleifera with exogenous application of plant growth regulators under saline conditions.

The study investigated the adaptability of Moringa oleifera to saline conditions, focusing on its hormesis behavior. It also examined how various plant growth regulators affected growth, physiological parameters, and bioactive compounds of moringa. In the first phase, different NaCl stress levels (0, 50, 100, 150, 200, and 250 mM) were applied. Notably, significant stimulation was observed at 100 mM stress for growth, total phenolics, total flavonoids and total chlorophyll content while 150 mM stress had a marked inhibitory effect, with survival decreasing at 200 and 250 mM NaCl levels. A 38% reduction in root attributes and shoot length, along with a 55% decrease in leaf score, was observed at 150 mM stress. Total phenolics showed a positive correlation with growth attributes. In the second phase, moringa plants grown under 50, 100, and 150 mM NaCl stress were treated with various plant growth regulators, including cytokinin (50 mg L-1), thiourea (5 mM), bezyl amino purine (BAP @50 mg L-1), salicylic acid (50 mg L-1), hydrogen peroxide (H2O2@120 µM), or ascorbic acid (50 mg L-1) to mitigate adverse effects of salinity. Cytokinin, BAP, and salicylic acid applications improved salinity tolerance, enhancing enzymatic, and non-enzymatic antioxidants, and the abundance of kaempferol, quercetin, hydroxybenzoic, and hydroxycinnamic acids. Pearson correlation and principal component analysis manifested relationships among growth parameters, antioxidant activities, flavonoids, and phenolic acids. This study provides new insights into hormesis management for moringa plants and the influence of plant growth regulators on flavonoids and phenolic acid levels in moringa leaves under saline conditions.

This study represents the first exploration of hormesis management in Moringa oleifera dual influence of changing soil conditions and foliar application of plant growth regulators. Additionally, this research fills a gap examining the variations in flavonoids (kaempferol and quercetin), hydroxycinnamic acids and hydroxybenzoic acids in moringa leaves concerning varying salinity levels and the exogenous application of plant growth regulators. Further, the study underscores the correlation among secondary metabolites, antioxidant activities and plant growth behavior.

Polymer brush grafted immobilized metal ion affinity adsorbent based on polydopamine/polyethyleneimine-coated magnetic graphene oxide for selective enrichment of cytokinins in plants.

An immobilized metal affinity (IMAC) adsorbent was prepared for selective enrichment of adenine type CKs, via grafting polymer chain pendant with iminodiacetic acid (IDA) from polydopamine (PDA)/polyethyleneimine (PEI)-coated magnetic graphene oxide (magGO) via surface-initiated-atom transfer radical polymerization (SI-ATRP). The prepared IMAC sorbent exhibited remarkable adsorption performances and good selectivity for adenine-type CKs and was utilized as a sorbent of magnetic solid-phase extraction (MSPE) for effective enrichment of four adenine-type CKs in bean sprouts. Under the optimized extraction conditions, an analytical method for four adenine type CKs in bean sprouts was established by combining the MSPE combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The recoveries of the analytes were between 80.4 ± 1.9% and 114.6 ± 1.5% (n = 3). The limits of detection (LODs) range from 0.63 to 2.30 pg⋅mL-1. The relative standard deviations of intra-day and inter-day were less than 12.6%. The established method was successfully applied to the selective extraction and sensitive detection of trace adenine-type CKs in plant samples.

A survey of Methylobacterium species and strains reveals widespread production and varying profiles of cytokinin phytohormones.

BACKGROUND: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). RESULTS: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL-1 for total CKs, and 0.46 to 82.16 pmol mL-1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. CONCLUSIONS: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations.

Phytohormone profiles in non-transformed and AtCKX transgenic centaury (Centaurium erythraea Rafn) shoots and roots in response to salinity stress in vitro.

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content.

Simultaneous quantitation of cytokinin bases and their glycoconjugates with stable isotope labelling ultrahigh performance liquid chromatography mass spectrometry.

Cytokinins (CKs) are one class of important phytohormones widely investigated in most aspects of plant life. Similar to other phytohormones, CKs and their glycoconjugates are hydrophilic. Their ionization efficiencies for mass spectrometry (MS) detection are rather poor, whereas their retention and separation on reverse phase liquid chromatography (RPLC) are often unsatisfying. Chemical isotope labelling LC-MS analysis methods have been developed for most other phytohormones, enhancing their LC separations and quantitative sensitivity. However, there are currently no reports for chemical-labelled CKs. Here, we report a new chemical isotope labelling LC-MS analytical method for one-pot derivatization of CK bases and their glycoconjugates, based on differential benzylation labelling of the adenine skeleton of CKs with benzyl bromide and its deuterium isotope-labelled reagent. Benzylation alters the hydrophilicity of CKs and their glycoconjugates, improving their retention and separation on RPLC. The developed method demonstrated enhanced sensitivity, as the CKs and their glycoconjugates could be analysed with LODs within the range of 0.62-25.9 pg/mL. The method also demonstrated good intra- and inter-day precisions with standard deviations in the range of 1.9%-13.0%, and acceptable accuracy with recoveries in the range of 84.0%-119.9%. The developed method was employed on the quantitation of CKs in the fresh roots of Astragalus membranaceus collected from both fertilized and unfertilized fields. The significant impact that fertilizers had on endogenous CKs metabolism was observed. As such, monitoring endogenous CKs and their metabolites might be promising to control fertilizer abuse.

Sensors for the quantification, localization and analysis of the dynamics of plant hormones.

Plant hormones play important roles in plant growth and development and physiology, and in acclimation to environmental changes. The hormone signaling networks are highly complex and interconnected. It is thus important to not only know where the hormones are produced, how they are transported and how and where they are perceived, but also to monitor their distribution quantitatively, ideally in a non-invasive manner. Here we summarize the diverse set of tools available for quantifying and visualizing hormone distribution and dynamics. We provide an overview over the tools that are currently available, including transcriptional reporters, degradation sensors, and luciferase and fluorescent sensors, and compare the tools and their suitability for different purposes.

Increased systemic IL-6 levels point to inflammation as a determinant of renal cell carcinoma development

Introduction: Renal cell carcinoma (RCC) is one of the most prevalent kidney tumors. Inflammation is believed to be a key factor in its progression and spread since inflammatory markers are generally associated with poor prognosis in RCC patients. Cytokines are cell communication molecules involved in both healthy and pathological processes, including tumor growth and progression. Recent findings suggest that cytokine level measurements could be used for cancer monitoring and prognosis. Methods: This study characterized and compared the levels of different cytokines associated with the classical Th1, Th2, and Th17 immune responses in plasma samples from RCC patients (n = 25) and healthy controls (n = 29). Cytokine levels (IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A) were evaluated by flow cytometry using a BD Cytometric Bead Array (CBA) kit. Results: No statistical differences in systemic IL-2, IL-4, IL-10, IL-17A, TNF, and INF-γ levels were observed between RCC patients and controls (p > 0.05). However, higher systemic IL-6 levels were observed in RCC patients (p = 0.0034). Conclusions: This study highlights the importance of assessing the impact of IL-6 on RCC pathogenesis and its potential role as a biomarker of disease progression. (AU)

Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

Transcription profiles reveal sugar and hormone signaling pathways mediating tree branch architecture in apple (Malus domestica Borkh.) grafted on different rootstocks.

Apple trees grafted on different rootstock types, including vigorous rootstock (VR), dwarfing interstock (DIR), and dwarfing self-rootstock (DSR), are widely planted in production, but the molecular determinants of tree branch architecture growth regulation induced by rootstocks are still not well known. In this study, the branch growth phenotypes of three combinations of 'Fuji' apple trees grafted on different rootstocks (VR: Malus baccata; DIR: Malus baccata/T337; DSR: T337) were investigated. The VR trees presented the biggest branch architecture. The results showed that the sugar content, sugar metabolism-related enzyme activities, and hormone content all presented obvious differences in the tender leaves and buds of apple trees grafted on these rootstocks. Transcriptomic profiles of the tender leaves adjacent to the top buds allowed us to identify genes that were potentially involved in signaling pathways that mediate the regulatory mechanisms underlying growth differences. In total, 3610 differentially expressed genes (DEGs) were identified through pairwise comparisons. The screened data suggested that sugar metabolism-related genes and complex hormone regulatory networks involved the auxin (IAA), cytokinin (CK), abscisic acid (ABA) and gibberellic acid (GA) pathways, as well as several transcription factors, participated in the complicated growth induction process. Overall, this study provides a framework for analysis of the molecular mechanisms underlying differential tree branch growth of apple trees grafted on different rootstocks.

Chemical composition of thermally processed coconut water evaluated by GC-MS, UPLC-HRMS, and NMR.

Thermally-processed coconut water often develop a commercially-undesirable pink color, thus, NMR, UPLC-HRMS, GC-MS analyses combined with chemometrics approach were applied to evaluate chemical variations in comparison to tender water (control) that could explain such color change. Chemometrics on negative ionization mode dataset showed trimeric and A-type dimeric procyanidins, and caffeoylshikimic acid as main identified secondary metabolites induced by processing, while, control water presented mainly cytokinin trans-zeatin riboside, procyanidin dimer, caffeoylshikimic acid and trihydroxy-octadecenoic acid. Processing increased long-chain saturated palmitic and stearic fatty acids contents, meanwhile NMR analysis showed a decline in primary metabolites content as sugars fructose and glucose, and short-chain organic acids. Among the results observed for thermally processed coconut water, the increase in oligomeric procyanidins as A-type dimer and trimer may be associated with pink color development as these are precursors of anthocyanin pigment and/or by enhancing color stability of anthocyanin solutions.

Life-Course Monitoring of Endogenous Phytohormone Levels under Field Conditions Reveals Diversity of Physiological States among Barley Accessions.

Agronomically important traits often develop during the later stages of crop growth as consequences of various plant-environment interactions. Therefore, the temporal physiological states that change and accumulate during the crop's life course can significantly affect the eventual phenotypic differences in agronomic traits among crop varieties. Thus, to improve productivity, it is important to elucidate the associations between temporal physiological responses during the growth of different crop varieties and their agronomic traits. However, data representing the dynamics and diversity of physiological states in plants grown under field conditions are sparse. In this study, we quantified the endogenous levels of five phytohormones - auxin, cytokinins (CKs), ABA, jasmonate and salicylic acid - in the leaves of eight diverse barley (Hordeum vulgare) accessions grown under field conditions sampled weekly over their life course to assess the ongoing fluctuations in hormone levels in the different accessions under field growth conditions. Notably, we observed enormous changes over time in the development-related plant hormones, such as auxin and CKs. Using 3' RNA-seq-based transcriptome data from the same samples, we investigated the expression of barley genes orthologous to known hormone-related genes of Arabidopsis throughout the life course. These data illustrated the dynamics and diversity of the physiological states of these field-grown barley accessions. Together, our findings provide new insights into plant-environment interactions, highlighting that there is cultivar diversity in physiological responses during growth under field conditions.

Endogenous levels of cytokinins, indole-3-acetic acid and abscisic acid in in vitro grown potato: A contribution to potato hormonomics.

A number of scientific reports published to date contain data on endogenous levels of various phytohormones in potato (Solanum tuberosum L.) but a complete cytokinin profile of potato tissues, that would include data on all particular molecular forms of cytokinin, has still been missing. In this work, endogenous levels of all analytically detectable isoprenoid cytokinins, as well as the auxin indole-3-acetic acid (IAA), and abscisic acid (ABA) have been determined in shoots and roots of 30 day old in vitro grown potato (cv. Désirée). The results presented here are generally similar to other data reported for in vitro grown potato plants, whereas greenhouse-grown plants typically contain lower levels of ABA, possibly indicating that in vitro grown potato is exposed to chronic stress. Cytokinin N-glucosides, particularly N7-glucosides, are the dominant cytokinin forms in both shoots and roots of potato, whereas nucleobases, as the bioactive forms of cytokinins, comprise a low proportion of cytokinin levels in tissues of potato. Differences in phytohormone composition between shoots and roots of potato suggest specific patterns of transport and/or differences in tissue-specific metabolism of plant hormones. These results represent a contribution to understanding the hormonomics of potato, a crop species of extraordinary economic importance.

Strategies for severe drought survival and recovery in a Pyrenean relict species.

In the context of future climate change new habitats will be threatened and unique species will be forced to develop different strategies to survive. Saxifraga longifolia Lapeyr. is an endemic species from the Pyrenees with a very particular habitat. We explored the capacity and strategies of S. longifolia plants to face different severities of drought stress under both natural conditions and controlled water stress followed by a re-watering period of 20 days. Our results showed a role for abscisic acid (ABA), salicylic acid (SA) and cytokinins (CKs) in plant survival from drought stress, and as the stress increased, ABA lost significance and SA appeared to be more associated with the response mechanisms. Moreover, photo-oxidative stress markers revealed that both xanthophyll cycles played a photoprotection role with a stronger participation of the lutein epoxide cycle as the stress was more intense. Severe drought decreased the maximum efficiency of photosystem II (Fv /Fm ) below 0.45, being this the limit to survive upon rewatering. Overall, our results proved different strategies of S. longifolia plants to cope with drought stress and suggested a Fv /Fm threshold to predict plant survival in high-mountain environments.

Natural variations in the promoter of Awn Length Inhibitor 1 (ALI-1) are associated with awn elongation and grain length in common wheat.

Wheat awn plays a vital role in photosynthesis, grain production, and drought tolerance. However, the systematic identification or cloning of genes controlling wheat awn development is seldom reported. Here, we conducted a genome-wide association study (GWAS) with 364 wheat accessions and identified 26 loci involved in awn length development, including previously characterized B1, B2, Hd, and several rice homologs. The dominant awn suppressor B1 was fine mapped to a 125-kb physical interval, and a C2 H2 zinc finger protein Awn Length Inhibitor 1 (ALI-1) was confirmed to be the underlying gene of the B1 locus through the functional complimentary test with native awnless allele. ALI-1 expresses predominantly in the developing spike of awnless individuals, transcriptionally suppressing downstream genes. ALI-1 reduces cytokinin content and simultaneously restrains cytokinin signal transduction, leading to a stagnation of cell proliferation and reduction of cell numbers during awn development. Polymorphisms of four single nucleotide polymorphisms (SNPs) located in ALI-1 promoter region are diagnostic for the B1/b1 genotypes, and these SNPs are associated with awn length (AL), grain length (GL) and thousand-grain weight (TGW). More importantly, ali-1 was observed to increase grain length in wheat, which is a valuable attribute of awn on grain weight, aside from photosynthesis. Therefore, ALI-1 pleiotropically regulates awn and grain development, providing an alternative for grain yield improvement and addressing future climate changes.

Quantification of Cytokinins Using High-Resolution Accurate-Mass Orbitrap Mass Spectrometry and Parallel Reaction Monitoring (PRM).

Cytokinins (CKs) are adenine derivatives that act as phytohormones. These signaling molecules control plant cell division and differentiation, organ growth, and senescence, and they orchestrate plant interactions with biotic and abiotic environments. While CKs are predominately recognized as plant-based substances, CKs have been found across different domains of life, including microorganisms, insects, mammals, and humans. In plants, CKs act at trace, often low femtomolar concentrations; therefore, sensitive and precise analytical techniques are required to accurately detect and quantify them from complex biological matrices. Here, we report the first comprehensive CK quantification method using a QExactive Orbitrap mass spectrometer in high-resolution with a parallel reaction monitoring (PRM)-based approach. The current method progresses upon multiple reaction monitoring (MRM) methods, previously used for CK profiling on triple quadrupole mass spectrometers. This method offers improved mass accuracy and the complete product ion mass spectra (MS/MS) for compound determination with increased specificity, and sensitivity comparable with triple quadrupole instruments. The presented PRM approach was successfully applied to quantify 32 CKs in several biological samples.

Direct electrochemistry of bacterial surface displayed cytokinin oxidase and its application in the sensitive electrochemical detection of cytokinins.

Cytokinin oxidase from Nipponbare (OsCKX4) was successfully displayed on the surface of E. coli cells by an ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif and a maltodextrin-binding protein(MBP) from E. coli as a solubility enhancer. The OsCKX4-displayed bacteria can be directly immobilized onto an electrode to selectively detect cytokinins, thus eliminating the need for enzyme extraction and purification. Direct electrochemistry of the cofactor FADH2 in OsCKX4 has been achieved on an edge-plane pyrolytic graphite electrode (PGE) with a formal potential (E0') of -0.45 V at pH 7.0 in phosphate buffer. With the addition of isopentenyladenine, the reduction peak current for FADH2 decreased, and the oxidative peak current increased gradually. Therefore, a bacteria-OsCKX4-modified PGE has been developed for the detection of isopentenyladenine with a linear range of 1.0-11.0 µM and a lower limit of detection of 0.7 µM (S/N = 3). Slight interference was observed in the presence of other phytohormones, including brassinosteroid, abscisic acid, methylene jasminate and gibberellin. The proposed bacterial biosensor is stable, specific and simple and has great potential for applications that require the detection of cytokinins.

Simultaneous determination of cytokinins by high performance liquid chromatography with resonance Rayleigh scattering and mechanism discussion.

A reliable, highly sensitive and highly selective method of high performance liquid chromatography associated with resonance Rayleigh scattering (HPLC-RRS) was developed to detect three cytokinins, namely, 6-benzylaminopurine (BA), kinetin (KT) and zeatin (ZT). In this work, Pd(ii) is added into the system to form ternary ion association complexes for the first time, which results in a lower limit of detection and extends the application of HPLC-RRS. The experimental conditions were optimized. In order to investigate the reaction mechanism, the ternary ion association complexes were characterized by ultraviolet-visible spectrophotometry, dynamic light scattering, scanning electron microscopy and density functional theory calculations. In a HAc-NaAc buffer solution (pH = 4.1), a ternary complex of cytokinin : Pd(ii) : EryB (1 : 1 : 2) was formed. The detection limits (S/N = 3) of BA, KT, and ZT were 0.9, 1.5 and 2.3 ng mL-1, respectively. In addition, this method was applied for the simultaneous detection of cytokinins in real samples with satisfactory results.

Transcriptome and hormone analyses provide insights into hormonal regulation in strawberry ripening.

MAIN CONCLUSION: The possible molecular mechanisms regulating strawberry fruit ripening were revealed by plant hormone quantification, exogenous hormone application, and RNA-sequencing. Fruit ripening involves a complex interplay among plant hormones. Strawberry is a model for studies on non-climacteric fruit ripening. However, the knowledge on how plant hormones are involved in strawberry ripening is still limited. To understand hormonal actions in the ripening process, we performed genome-wide transcriptome and hormonal analysis for the five major hormones (abscisic acid and catabolites, auxins, cytokinins, gibberellins, and ethylene) in achenes and receptacles (flesh) at different ripening stages of the woodland strawberry Fragaria vesca. Our results demonstrate that the pre-turning stage (a stage with white flesh and red achenes defined in this study) is the transition stage from immature to ripe fruits. The combinatorial analyses of hormone content, transcriptome data, and exogenous hormone treatment indicate that auxin is synthesized predominantly in achenes, while abscisic acid (ABA), bioactive free base cytokinins, gibberellins, and ethylene are mainly produced in receptacles. Furthermore, gibberellin may delay ripening, while ethylene and cytokinin are likely involved at later stages of the ripening process. Our results also provide additional evidence that ABA promotes ripening, while auxin delays it. Although our hormone analysis demonstrates that the total auxin in receptacles remains relatively low and unchanged during ripening, our experimental evidence further indicates that ABA likely enhances expression of the endoplasmic reticulum-localized auxin efflux carrier PIN-LIKES, which may subsequently reduce the auxin level in nucleus. This study provides a global picture for hormonal regulation of non-climacteric strawberry fruit ripening and also evidence for a possible mechanism of ABA and auxin interaction in the ripening process.

Hormonal Responses to Plasmodiophora brassicae Infection in Brassica napus Cultivars Differing in Their Pathogen Resistance.

Hormonal dynamics after Plasmodiophora brassicae infection were compared in two Brassica napus cultivars-more resistant SY Alister and more sensitive Hornet, in order to elucidate responses associated with efficient defense. Both cultivars responded to infection by the early transient elevation of active cytokinins (predominantly cis-zeatin) and auxin indole-3-acetic acid (IAA) in leaves and roots, which was longer in Hornet. Moderate IAA levels in Hornet roots coincided with a high expression of biosynthetic gene nitrilase NIT1 (contrary to TAA1, YUC8, YUC9). Alister had a higher basal level of salicylic acid (SA), and it stimulated its production (via the expression of isochorismate synthase (ICS1)) in roots earlier than Hornet. Gall formation stimulated cytokinin, auxin, and SA levels-with a maximum 22 days after inoculation (dai). SA marker gene PR1 expression was the most profound at the time point where gall formation began, in leaves, roots, and especially in galls. Jasmonic acid (JA) was higher in Hornet than in Alister during the whole experiment. To investigate SA and JA function, SA was applied before infection, and twice (before infection and 15 dai), and JA at 15 dai. Double SA application diminished gall formation in Alister, and JA promoted gall formation in both cultivars. Activation of SA/JA pathways reflects the main differences in clubroot resistance.

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry.

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.