Phytohormone profiling in an evolutionary framework.

The genomes of charophyte green algae, close relatives of land plants, typically do not show signs of developmental regulation by phytohormones. However, scattered reports of endogenous phytohormone production in these organisms exist. We performed a comprehensive analysis of multiple phytohormones in Viridiplantae, focusing mainly on charophytes. We show that auxin, salicylic acid, ethylene and tRNA-derived cytokinins including cis-zeatin are found ubiquitously in Viridiplantae. By contrast, land plants but not green algae contain the trans-zeatin type cytokinins as well as auxin and cytokinin conjugates. Charophytes occasionally produce jasmonates and abscisic acid, whereas the latter is detected consistently in land plants. Several phytohormones are excreted into the culture medium, including auxin by charophytes and cytokinins and salicylic acid by Viridiplantae in general. We note that the conservation of phytohormone biosynthesis and signaling pathways known from angiosperms does not match the capacity for phytohormone biosynthesis in Viridiplantae. Our phylogenetically guided analysis of established algal cultures provides an important insight into phytohormone biosynthesis and metabolism across Streptophyta.

Uncovering the Interrelation between Metabolite Profiles and Bioactivity of In Vitro- and Wild-Grown Catmint (Nepeta nuda L.).

Nepeta nuda L. is a medicinal plant enriched with secondary metabolites serving to attract pollinators and deter herbivores. Phenolics and iridoids of N. nuda have been extensively investigated because of their beneficial impacts on human health. This study explores the chemical profiles of in vitro shoots and wild-grown N. nuda plants (flowers and leaves) through metabolomic analysis utilizing gas chromatography and mass spectrometry (GC-MS). Initially, we examined the differences in the volatiles' composition in in vitro-cultivated shoots comparing them with flowers and leaves from plants growing in natural environment. The characteristic iridoid 4a-α,7-ß,7a-α-nepetalactone was highly represented in shoots of in vitro plants and in flowers of plants from nature populations, whereas most of the monoterpenes were abundant in leaves of wild-grown plants. The known in vitro biological activities encompassing antioxidant, antiviral, antibacterial potentials alongside the newly assessed anti-inflammatory effects exhibited consistent associations with the total content of phenolics, reducing sugars, and the identified metabolic profiles in polar (organic acids, amino acids, alcohols, sugars, phenolics) and non-polar (fatty acids, alkanes, sterols) fractions. Phytohormonal levels were also quantified to infer the regulatory pathways governing phytochemical production. The overall dataset highlighted compounds with the potential to contribute to N. nuda bioactivity.

Accumulation of Toxic Arsenic by Cherry Radish Tuber (Raphanus sativus var. sativus Pers.) and Its Physiological, Metabolic and Anatomical Stress Responses.

In a pot experiment, cherry radish (Raphanus sativus var. sativus Pers. 'Viola') was cultivated under two levels of As soil contamination-20 and 100 mg/kg. The increasing As content in tubers with increasing soil contamination led to changes in free amino acids (AAs) and phytohormone metabolism and antioxidative metabolites. Changes were mainly observed under conditions of high As contamination (As100). The content of indole-3-acetic acid in tubers varied under different levels of As stress, but As100 contamination led to an increase in its bacterial precursor indole-3-acetamide. A decrease in cis-zeatin-9-riboside-5'-monophosphate content and an increase in jasmonic acid content were found in this treatment. The free AA content in tubers was also reduced. The main free AAs were determined to be transport AAs (glutamate-Glu, aspartate, glutamine-Gln, asparagine) with the main portion being Gln. The Glu/Gln ratio-a significant indicator of primary N assimilation in plants-decreased under the As100 treatment condition. A decrease in antioxidative metabolite content-namely that of ascorbic acid and anthocyanins-was observed in this experiment. A decline in anthocyanin content is related to a decrease in aromatic AA content which is crucial for secondary metabolite production. The changes in tubers caused by As contamination were reflected in anatomical changes in the radish tubers and roots.

Comprehensive profiling of endogenous phytohormones and expression analysis of 1-aminocyclopropane-1-carboxylic acid synthase gene family during fruit development and ripening in octoploid strawberry (Fragaria× ananassa).

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(Δ2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N6-(Δ2-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N6-(Δ2-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

What determines root-sprouting ability: Injury or phytohormones?

PREMISE: Root-sprouting (RS) is an evolutionarily independent alternative to axillary stem branching for a plant to attain its architecture. Root-sprouting plants are better adapted to disturbance than non-RS plants, and their vigor is frequently boosted by biomass removal. Nevertheless, RS plants are rarer than plants that are not root-sprouters, possibly because they must overcome developmental barriers such as intrinsic phytohormonal balance or because RS ability is conditioned by injury to the plant body. The objective of this study was to identify whether phytohormones or injury enable RS. METHODS: In a greenhouse experiment, growth variables, root respiration, and phytohormones were analyzed in two closely related clonal herbs that differ in RS ability (spontaneously RS Inula britannica and rhizomatous non-RS I. salicina) with and without severe biomass removal. RESULTS: As previously reported, I. britannica is a root-sprouter, but injury did not boost its RS ability. Root respiration did not differ between the two species and decreased continuously with time irrespectively of injury, but their phytohormone profiles differed significantly. In RS species, the auxins-to-cytokinins ratio was low, and injury further decreased it. CONCLUSIONS: This first attempt to test drivers behind different plant growth forms suggests that intrinsic phytohormone regulation, especially the auxins-to-cytokinins ratio, might be behind RS ability. Injury, causing a phytohormonal imbalance, seems to be less important in spontaneously RS species than expected for RS species in general.

Cytokinin deficiency confers enhanced tolerance to mild, but decreased tolerance to severe salinity stress in in vitro grown potato.

Cytokinin (CK) is a plant hormone that plays crucial roles in regulating plant growth and development. CK-deficient plants are widely used as model systems for investigating the numerous physiological roles of CK. Since it was previously shown that transgenic or mutant CK-deficient Arabidopsis and Centaurium plants show superior tolerance to salinity, we examined the tolerance of three CK-deficient potato lines overexpressing the Arabidopsis thaliana CYTOKININ OXIDASE/DEHYDROGENASE2 (AtCKX2) gene to 50 mM, 100 mM, 150 mM, and 200 mM NaCl applied in vitro. Quantification of visible salinity injury, rooting and acclimatization efficiency, shoot growth, water saturation deficit, and chlorophyll content confirmed that the CK-deficient potato plants were more tolerant to low (50 mM) and moderate (100 mM) NaCl concentrations, but exhibited increased sensitivity to severe salinity stress (150 and 200 mM NaCl) compared to non-transformed control plants. These findings were corroborated by the data distribution patterns according to principal component analysis. Quantification of the activity of superoxide dismutases, peroxidases, and catalases revealed an impaired ability of AtCKX2-transgenic lines to upregulate the activity of antioxidant enzymes in response to salinity, which might contribute to the enhanced sensitivity of these potato lines to severe salt stress. Our results add complexity to the existing knowledge on the regulation of salinity tolerance by CK, as we show for the first time that CK-deficient plants can exhibit reduced rather than increased tolerance to severe salt stress.

Evolutionary Aspects of Hypericin Productivity and Endogenous Phytohormone Pools Evidenced in Hypericum Species In Vitro Culture Model.

Shoot cultures of hypericin non-producing H. calycinum L. (primitive Ascyreia section), hypericin-producing H. perforatum L., H. tetrapterum Fries (section Hypericum) and H. richeri Vill. (the evolutionarily most advanced section Drosocarpium in our study) were developed and investigated for their growth, development, hypericin content and endogenous phytohormone levels. Hypericins in wild-growing H. richeri significantly exceeded those in H. perforatum and H. tetrapterum. H. richeri also had the highest hypericin productivity in vitro in medium supplemented with 0.2 mg/L N6-benzyladenine and 0.1 mg/L indole-3-butyric acid and H. tetrapterum-the lowest one in all media modifications. In shoot culture conditions, the evolutionarily oldest H. calycinum had the highest content of salicylic acid and total jasmonates in some of its treatments, as well as dominance of the storage form of abscisic acid (ABA-glucose ester) and lowest cytokinin ribosides and cytokinin O-glucosides as compared with the other three species. In addition, the evolutionarily youngest H. richeri was characterized by the highest total amount of cytokinin ribosides. Thus, both evolutionary development and the hypericin production capacity seemed to interact closely with the physiological parameters of the plant organism, such as endogenous phytohormones, leading to the possible hypothesis that hypericin productivity may have arisen in the evolution of Hypericum as a means to adapt to environmental changes.

Comprehensive Phytohormone Profiling of Kohlrabi during In Vitro Growth and Regeneration: The Interplay with Cytokinin and Sucrose.

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1-T4), additionally including the effects of cytokinins (CKs)-trans-zeatin (transZ) and thidiazuron (TDZ)-and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses.

The humidity level matters during the desiccation of Norway spruce somatic embryos.

In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes ß-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some ß-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

Periodicity and Spectral Composition of Light in the Regulation of Hypocotyl Elongation of Sunflower Seedlings.

This study presents the hypocotyl elongation of sunflower seedlings germinated under different light conditions. Elongation was rhythmic under diurnal (LD) photoperiods but uniform (arrhythmic) under free-running conditions of white light (LL) or darkness (DD). On the sixth day after the onset of germination, seedlings were entrained in all diurnal photoperiods. Their hypocotyl elongation was dual, showing different kinetics in daytime and nighttime periods. The daytime elongation peak was around midday and 1-2 h after dusk in the nighttime. Plantlets compensated for the differences in the daytime and nighttime durations and exhibited similar overall elongation rates, centered around the uniform elongation in LL conditions. Thus, plants from diurnal photoperiods and LL could be grouped together as white-light treatments that suppressed hypocotyl elongation. Hypocotyl elongation was significantly higher under DD than under white-light photoperiods. In continuous monochromatic blue, yellow, green, or red light, hypocotyl elongation was also uniform and very high. The treatments with monochromatic light and DD had similar overall elongation rates; thus, they could be grouped together. Compared with white light, monochromatic light promoted hypocotyl elongation. Suppression of hypocotyl elongation and rhythmicity reappeared in some combination with two or more monochromatic light colors. The presence of red light was obligatory for this suppression. Plantlets entrained in diurnal photoperiods readily slipped from rhythmic into uniform elongation if they encountered any kind of free-running conditions. These transitions occurred whenever the anticipated duration of daytime or nighttime was extended more than expected, or when plantlets were exposed to constant monochromatic light. This study revealed significant differences in the development of sunflower plantlets illuminated with monochromatic or white light.

Desiccation as a Post-maturation Treatment Helps Complete Maturation of Norway Spruce Somatic Embryos: Carbohydrates, Phytohormones and Proteomic Status.

Exposure of Norway spruce (Picea abies) somatic embryos and those of many other conifers to post-maturation desiccation treatment significantly improves their germination. An integration analysis was conducted to understand the underlying processes induced during the desiccation phase at the molecular level. Carbohydrate, protein and phytohormone assays associated with histological and proteomic studies were performed for the evaluation of markers and actors in this phase. Multivariate comparison of mature somatic embryos with mature desiccated somatic embryos and/or zygotic embryos provided new insights into the processes involved during the desiccation step of somatic embryogenesis. Desiccated embryos were characterized by reduced levels of starch and soluble carbohydrates but elevated levels of raffinose family oligosaccharides. Desiccation treatment decreased the content of abscisic acid and its derivatives but increased total auxins and cytokinins. The content of phytohormones in dry zygotic embryos was lower than in somatic embryos, but their profile was mostly analogous, apart from differences in cytokinin profiles. The biological processes "Acquisition of desiccation tolerance", "Response to stimulus", "Response to stress" and "Stored energy" were activated in both the desiccated somatic embryos and zygotic embryos when compared to the proteome of mature somatic embryos before desiccation. Based on the specific biochemical changes of important constituents (abscisic acid, raffinose, stachyose, LEA proteins and cruciferins) induced by the desiccation treatment and observed similarities between somatic and zygotic P. abies embryos, we concluded that the somatic embryos approximated to a state of desiccation tolerance. This physiological change could be responsible for the reorientation of Norway spruce somatic embryos toward a stage suitable for germination.

Hormonome Dynamics During Microgametogenesis in Different Nicotiana Species.

Plant microgametogenesis involves stages leading to the progressive development of unicellular microspores into mature pollen. Despite the active and continuing interest in the study of male reproductive development, little is still known about the hormonomics at each ontogenetic stage. In this work, we characterized the profiles and dynamics of phytohormones during the process of microgametogenesis in four Nicotiana species (Nicotiana tabacum, Nicotiana alata, Nicotiana langsdorffii, and Nicotiana mutabilis). Taking advantage of advanced HPLC-ESI-MS/MS, twenty to thirty endogenous hormone derivatives were identified throughout pollen ontogenesis, including cytokinins, auxins, ABA and its derivatives, jasmonates, and phenolic compounds. The spectra of endogenous phytohormones changed dynamically during tobacco pollen ontogeny, indicating their important role in pollen growth and development. The different dynamics in the accumulation of endogenous phytohormones during pollen ontogenesis between N. tabacum (section Nicotiana) and the other three species (section Alatae) reflects their different phylogenetic positions and origin within the genus Nicotiana. We demonstrated the involvement of certain phytohormone forms, such as cis-zeatin- and methylthiol-type CKs, some derivatives of abscisic acid, phenylacetic and benzoic acids, in pollen development for the first time here. Our results suggest that unequal levels of endogenous hormones and the presence of specific derivatives may be characteristic for pollen development in different phylogenetic plant groups. These results represent the currently most comprehensive study of plant hormones during the process of pollen development.

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.

Integrating the Roles for Cytokinin and Auxin in De Novo Shoot Organogenesis: From Hormone Uptake to Signaling Outputs.

De novo shoot organogenesis (DNSO) is a procedure commonly used for the in vitro regeneration of shoots from a variety of plant tissues. Shoot regeneration occurs on nutrient media supplemented with the plant hormones cytokinin (CK) and auxin, which play essential roles in this process, and genes involved in their signaling cascades act as master regulators of the different phases of shoot regeneration. In the last 20 years, the genetic regulation of DNSO has been characterized in detail. However, as of today, the CK and auxin signaling events associated with shoot regeneration are often interpreted as a consequence of these hormones simply being present in the regeneration media, whereas the roles for their prior uptake and transport into the cultivated plant tissues are generally overlooked. Additionally, sucrose, commonly added to the regeneration media as a carbon source, plays a signaling role and has been recently shown to interact with CK and auxin and to affect the efficiency of shoot regeneration. In this review, we provide an integrative interpretation of the roles for CK and auxin in the process of DNSO, adding emphasis on their uptake from the regeneration media and their interaction with sucrose present in the media to their complex signaling outputs that mediate shoot regeneration.

Evolutionary diversification of cytokinin-specific glucosyltransferases in angiosperms and enigma of missing cis-zeatin O-glucosyltransferase gene in Brassicaceae.

In the complex process of homeostasis of phytohormones cytokinins (CKs), O-glucosylation catalyzed by specific O-glucosyltransferases represents one of important mechanisms of their reversible inactivation. The CK O-glucosyltransferases belong to a highly divergent and polyphyletic multigene superfamily of glycosyltransferases, of which subfamily 1 containing UDP-glycosyltransferases (UGTs) is the largest in the plant kingdom. It contains recently discovered O and P subfamilies present in higher plant species but not in Arabidopsis thaliana. The cis-zeatin O-glucosyltransferase (cisZOG) genes belong to the O subfamily encoding a stereo-specific O-glucosylation of cis-zeatin-type CKs. We studied different homologous genes, their domains and motifs, and performed a phylogenetic reconstruction to elucidate the plant evolution of the cisZOG gene. We found that the cisZOG homologs do not form a clear separate clade, indicating that diversification of the cisZOG gene took place after the diversification of the main angiosperm families, probably within genera or closely related groups. We confirmed that the gene(s) from group O is(are) not present in A. thaliana and is(are) also missing in the family Brassicaceae. However, cisZOG or its metabolites are found among Brassicaceae clade, indicating that remaining genes from other groups (UGT73-group D and UGT85-group G) are able, at least in part, to substitute the function of group O lost during evolution. This study is the first detailed evolutionary evaluation of relationships among different plant ZOGs within angiosperms.

Sucrose interferes with endogenous cytokinin homeostasis and expression of organogenesis-related genes during de novo shoot organogenesis in kohlrabi.

Cross-talk between phytohormones and sugars is intensely involved in plant metabolism, growth and regeneration. We documented alterations in cytokinin (CK) homeostasis in four developmental stages during de novo shoot organogenesis (DNSO) of kohlrabi (Brassica oleracea var. gongylodes cv. Vienna Purple) seedlings induced by exogenous CKs, trans-zeatin (transZ) and thidiazuron (TDZ), added together with elevated sucrose concentration (6% and 9%). Significant impact of CK and sucrose treatment and their interaction was recorded in all investigated stages, including plantlet development before calli formation (T1 and T2), calli formation (T3) and shoot regeneration (T4). Results showed remarkable increase in total CK levels for transZ treatment, particularly with 9% sucrose. This trend was observed for all physiological and structural groups of CKs. Application of TDZ contributed to little or no increase in CK levels regardless of sucrose concentration. Analysis of expression profiles of organogenesis-related genes involved in auxin transport, CK response, shoot apical meristem formation and cell division revealed that higher sugar concentration significantly downregulated the analysed genes, particularly in T3. This continued on TDZ, but transZ induced an opposite effect with 9% sucrose in T4, increasing gene activity. Our results demonstrated that phytohormone metabolism might be triggered by sucrose signalling in kohlrabi DNSO.

Heat Stress Targeting Individual Organs Reveals the Central Role of Roots and Crowns in Rice Stress Responses.

Inter-organ communication and the heat stress (HS; 45°C, 6 h) responses of organs exposed and not directly exposed to HS were evaluated in rice (Oryza sativa) by comparing the impact of HS applied either to whole plants, or only to shoots or roots. Whole-plant HS reduced photosynthetic activity (F v /F m and QY_Lss ), but this effect was alleviated by prior acclimation (37°C, 2 h). Dynamics of HSFA2d, HSP90.2, HSP90.3, and SIG5 expression revealed high protection of crowns and roots. Additionally, HSP26.2 was strongly expressed in leaves. Whole-plant HS increased levels of jasmonic acid (JA) and cytokinin cis-zeatin in leaves, while up-regulating auxin indole-3-acetic acid and down-regulating trans-zeatin in leaves and crowns. Ascorbate peroxidase activity and expression of alternative oxidases (AOX) increased in leaves and crowns. HS targeted to leaves elevated levels of JA in roots, cis-zeatin in crowns, and ascorbate peroxidase activity in crowns and roots. HS targeted to roots increased levels of abscisic acid and auxin in leaves and crowns, cis-zeatin in leaves, and JA in crowns, while reducing trans-zeatin levels. The weaker protection of leaves reflects the growth strategy of rice. HS treatment of individual organs induced changes in phytohormone levels and antioxidant enzyme activity in non-exposed organs, in order to enhance plant stress tolerance.

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants.

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.

CgIPT1 is required for synthesis of cis-zeatin cytokinins and contributes to stress tolerance and virulence in Colletotrichum graminicola.

We have previously shown that the maize pathogen Colletotrichum graminicola is able to synthesise cytokinins (CKs). However, it remained unsettled whether fungal CK production is essential for virulence in this hemibiotrophic fungus. Here, we identified a candidate gene, CgIPT1, that is homologous to MOD5 of Saccharomyces cerevisiae and genes from other fungi and plants, which encode tRNA-isopentenyltransferases (IPTs). We show that the wild type strain mainly synthesises cis-zeatin-type (cisZ) CKs whereas ΔCgipt1 mutants are severely impeded to do so. The spectrum of CKs produced confirms bioinformatical analyses predicting that CgIpt1 is a tRNA-IPT. The virulence of the ΔCgipt1 mutants is moderately reduced. Furthermore, the mutants exhibit increased sensitivities to osmotic stress imposed by sugar alcohols and salts, as well as cell wall stress imposed by Congo red. Amendment of media with CKs did not reverse this phenotype suggesting that fungal-derived CKs do not explain the role of CgIpt1 in mediating abiotic stress tolerance. Moreover, the mutants still cause green islands on senescing maize leaves indicating that the cisZ-type CKs produced by the fungus do not cause this phenotype.

Response of cytokinins and nitrogen metabolism in the fronds of Pteris sp. under arsenic stress.

Given the close relationship between cytokinins (CKs), photosynthesis and nitrogen metabolism, this study assessed the effect of arsenic (As) contamination on these metabolic components in the As-hyperaccumulators Pteris cretica L. var. Albo-lineata (Pc-A) and var. Parkerii (Pc-P) as well as the As-non-hyperaccumulator Pteris straminea Mett. ex Baker (Ps). The ferns were cultivated in a pot experiment for 23 weeks in soil spiked with As at the levels 20 and 100 mg·kg-1. For the purpose of this study, the CKs were placed into five functionally different groups according to their structure and physiological roles: bioactive forms (bCKs; CK free bases); inactive or weakly active forms (dCKs; CK N-glucosides); transport forms (tCKs; CK ribosides); storage forms (sCKs; O-glucosides); and primary products of CK biosynthesis (ppbCKs; CK nucleotides). An important finding was higher CKs total content, accumulation of sCKs and reduction of dCKs in As-hyperaccumulators in contrast to non-hyperaccumulator ferns. A significant depletion of C resources was confirmed in ferns, especially Ps, which was determined by measuring the photosynthetic rate and chlorophyll fluorescence. A fluorescence decrease signified a reduction in the C/N ratio, inducing an increase of bioactive CKs forms in Pc-P and Ps. The impact of As on N utilization was significant in As-hyperaccumulators. The glutamic acid/glutamine ratio, an indicator of primary N assimilation, diminished in all ferns with increased As level in the soil. In conclusion, the results indicate a large phenotypic diversity of Pteris species to As and suggest that the CKs composition and the glutamic acid/glutamine ratio can be used as a tool to diagnose As stress in plants.