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.

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.

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.

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

The Response of Picea abies Somatic Embryos to UV-B Radiation Depends on the Phase of Maturation.

Ultraviolet-B (UV-B) radiation is a key environmental signal which initiates diverse responses that affect the metabolism, development, and viability of plants. In keeping with our previous studies, we concentrated primarily on how UV-B radiation affects Norway spruce [Picea abies (L.) Karst.] somatic embryo maturation and how phenolics and polyamines (PAs) are linked to the defense response invoked by UV-B irradiation. We treated clusters of Norway spruce embryogenic culture (EC) with UV-B during the five stages of embryo maturation (early, cylindrical, precotyledonary, cotyledonary, and mature embryos). For the first time, we take an advantage of the unique environmental scanning electron microscope AQUASEM II to characterize somatic embryos in their native state. The severity of the irradiation effect on embryonal cell viability was shown to be dependent on the intensity of radiation as well as the stage of embryo development, and might be related to the formation of protoderm. The response of early embryos was characterized by an increase in malondialdehyde (MDA), a marked decrease in PA contents and a decline in phenolics. The reduced ability to activate the defense system seems to be responsible not only for the severe cell damage and decrease in viability but also for the inhibition of embryo development. The significant reduction in spermidine (Spd), which has been reported to be crucial for the somatic embryo development of several coniferous species, may be causally linked to the limited development of embryos. The pronounced decrease in cell wall-bound ferulic acid might correspond to failure of somatic embryos to reach more advanced stages of development. Embryos at later stages of development showed stress defense responses that were more efficient against UV-B exposure.

Tobacco susceptibility to Potato virus Y(NTN) infection is affected by grafting and endogenous cytokinin content.

Faster or stronger response to pathogen occurs if plants undergo prior priming. Cytokinins seem to be also involved in plant priming and in response to pathogens. Susceptibility to Potato virus Y(NTN) (PVY(NTN)) was studied in transgenic cytokinin overproducing (Pssu-ipt) tobacco and compared with nontransgenic plants. Since cytokinin overproduction inhibits development of plant roots and grafting overcomes this limitation, both types were grown as rooted and/or grafted plants to check also the effect of grafting. Control rooted tobacco (C), the most susceptible to PVY(NTN), showed always symptoms during the infection together with the rising virus content and a systemic response, such as accumulation of H2O2, salicylic acid (SA) and other phenolic acids, and stress-induced enzyme activities. In transgenic and grafted plants, the response to PVY(NTN) was dependent on protective mechanisms activated prior to the inoculation. In Pssu-ipt tobacco, cytokinin active forms and SA contents exceeded manifold their content in C. Grafting promoted the accumulation of phenolics, but SA, and stimulated peroxidase activities. Thus, the pre-infection barrier established in both transgenic and grafted plants helped to suppress partly the virus multiplication and resulted in milder symptom development. However, only the synergic effect of both grafting and the high cytokinins led to PVY(NTN) tolerance in transgenic grafts. Possible mechanisms were discussed.

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis.

Targeting of the heat stress (HS, 40°C) to shoots, roots or whole plants substantially affects Arabidopsis physiological responses. Effective stress targeting was proved by determination of the expression of HS markers, HsfA2 and HSA32, which were quickly stimulated in the targeted organ(s), but remained low in non-stressed tissues for at least 2h. When shoots or whole plants were subjected to HS, a transient decrease in abscisic acid, accompanied by a small increase in active cytokinin levels, was observed in leaves, consistent with stimulation of transpiration, the main cooling mechanism in leaves. HS application targeted to part of plant resulted in a rapid stimulation of expression of components of cytokinin signaling pathway (especially of receptor genes) in the non-exposed tissues, which indicated fast inter-organ communication. Under all HS treatments, shoot apices responded by transient elevation of active cytokinin contents and stimulation of transcription of genes involved in photosynthesis and carbohydrate metabolism. Duration of this stimulation was negatively correlated with stress strength. The impact of targeted HS on the expression of 63 selected genes, including those coding regulatory 14-3-3 proteins, was compared. Stimulation of GRF9 (GRF14µ) in stressed organs after 2-6h may be associated with plant stress adaptation.

Proteome analysis in Arabidopsis reveals shoot- and root-specific targets of cytokinin action and differential regulation of hormonal homeostasis.

The plant hormones cytokinins (CKs) regulate multiple developmental and physiological processes in Arabidopsis (Arabidopsis thaliana). Responses to CKs vary in different organs and tissues (e.g. the response to CKs has been shown to be opposite in shoot and root samples). However, the tissue-specific targets of CKs and the mechanisms underlying such specificity remain largely unclear. Here, we show that the Arabidopsis proteome responds with strong tissue and time specificity to the aromatic CK 6-benzylaminopurine (BAP) and that fast posttranscriptional and/or posttranslational regulation of protein abundance is involved in the contrasting shoot and root proteome responses to BAP. We demonstrate that BAP predominantly regulates proteins involved in carbohydrate and energy metabolism in the shoot as well as protein synthesis and destination in the root. Furthermore, we found that BAP treatment affects endogenous hormonal homeostasis, again with strong tissue specificity. In the shoot, BAP up-regulates the abundance of proteins involved in abscisic acid (ABA) biosynthesis and the ABA response, whereas in the root, BAP rapidly and strongly up-regulates the majority of proteins in the ethylene biosynthetic pathway. This was further corroborated by direct measurements of hormone metabolites, showing that BAP increases ABA levels in the shoot and 1-aminocyclopropane-1-carboxylic acid, the rate-limiting precursor of ethylene biosynthesis, in the root. In support of the physiological importance of these findings, we identified the role of proteins mediating BAP-induced ethylene production, METHIONINE SYNTHASE1 and ACC OXIDASE2, in the early root growth response to BAP.

Remodeling of cytokinin metabolism at infection sites of Colletotrichum graminicola on maize leaves.

When inoculated onto maize leaves at the onset of senescence, the hemibiotroph Colletotrichum graminicola causes green islands that are surrounded by senescing tissue. Taking advantage of green islands as indicators of sites of the establishment of successful infection and of advanced high-performance liquid chromatography tandem mass spectrometry methodology, we analyzed changes in the patterns and levels of cytokinins (CK) at high spatial and analytical resolution. Twenty individual CK were detected in green islands. Levels of cis-zeatin-9-riboside and cis-zeatin-9-riboside-5'-monophosphate increased drastically, whereas that of the most prominent CK, cis-zeatin-O-glucoside, decreased. The fungus likely performed these conversions because corresponding activities were also detected in in vitro cultures amended with CK. We found no evidence that C. graminicola is able to synthesize CK entirely de novo in minimal medium but, after adding dimethylallyl diphosphate, a precursor of CK biosynthesis occurring in plants, a series of trans-zeatin isoforms (i.e., trans-zeatin-9-riboside-5'-monophosphate, trans-zeatin-9-riboside, and trans-zeatin) was formed. After applying CK onto uninfected leaves, transcripts of marker genes for senescence, photosynthesis, and assimilate distribution were measured by quantitative reverse-transcribed polymerase chain reaction; furthermore, pulse-amplitude modulation chlorophyll fluorometry and single-photon avalanche diode analyses were conducted. These experiments suggested that modulation of CK metabolism at the infection site affects host physiology.

Retargeting a maize ß-glucosidase to the vacuole--evidence from intact plants that zeatin-O-glucoside is stored in the vacuole.

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize ß-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

Comparison of hormonal responses to heat, drought and combined stress in tobacco plants with elevated proline content.

In order to test the possibility of improving tolerance to heat and drought (alone and in combination) by elevation of the osmoprotectant proline (Pro) content, stress responses were compared in tobacco plants constitutively over-expressing a gene for the Pro biosynthetic enzyme Δ(2)-pyrroline-5-carboxylate synthetase (P5CSF129A; EC 2.7.2.11/1.2.1.41) and in the corresponding wild-type. Significantly enhanced Pro production in the transformant coincided with a more negative leaf osmotic potential (both at control conditions and following stress) and enhanced production of protective xanthophyll cycle pigments. Heat stress (40 °C) caused a transient increase in the level of bioactive cytokinins, predominantly N(6)-(2-isopentenyl)adenosine, accompanied by down-regulation of the activity of the main cytokinin degrading enzyme cytokinin oxidase/dehydrogenase (EC 1.4.3.18/1.5.99.12). No significant differences were found between the tested genotypes. In parallel, a transient decrease of abscisic acid was observed. Following drought stress, bioactive cytokinin levels decreased in the whole plants, remaining relatively higher in preferentially protected upper leaves and in roots. Cytokinin suppression was less pronounced in Pro transformants. Exposure to heat stress (40 °C for 2h) at the end of 10-d drought period strongly enhanced the severity of the water stress, as indicated by a drop in leaf water potential. The activity of cytokinin oxidase/dehydrogenase was strongly stimulated in upper leaves and roots of stressed plants, coinciding with strong down-regulation of bioactive cytokinins in whole plants. Combined heat and drought stress resulted in a minor decrease in abscisic acid, but only in non-wilty upper leaves. Both stresses as well as their combination were associated with elevation of free auxin, indolylacetic acid, in lower leaves and/or in roots. Auxin increase was dependent on the stress strength. After rehydration, a marked elevation of bioactive cytokinins in leaves was observed. A greater increase in cytokinin content in Pro transformants indicated a mild elevation of their stress tolerance.

AtSOFL1 and AtSOFL2 act redundantly as positive modulators of the endogenous content of specific cytokinins in Arabidopsis.

BACKGROUND: Although cytokinins have been known for decades to play important roles in the regulation of plant growth and development, our knowledge of the regulatory mechanism of endogenous content of specific cytokinins remains limited. METHODOLOGY/PRINCIPAL FINDINGS: Here, we characterized two SOB five-like (SOFL) genes, AtSOFL1 and AtSOFL2, in Arabidopsis (Arabidopsis thaliana) and showed that they acted redundantly in regulating specific cytokinin levels. Analysis of the translational fusion AtSOFL1:AtSOFL1-GUS and AtSOFL2:AtSOFL2-GUS indicated that AtSOFL1 and AtSOFL2 exhibited similar expression patterns. Both proteins were predominantly expressed in the vascular tissues of developing leaves, flowers and siliques, but barely detectable in roots and stems. Overexpression of either AtSOFL1 or AtSOFL2 led to increased cytokinin content and obvious corresponding mutant phenotypes for both transgenic seedlings and adult plants. In addition, overexpression and site-directed mutagenesis experiments demonstrated that the SOFL domains are necessary for AtSOFL2's overexpression phenotypes. Silencing or disrupting either AtSOFL1 or AtSOFL2 caused no obvious developmental defects. Endogenous cytokinin analysis, however, revealed that compared to the wild type control, the SOFL1-RNAi62 sofl2-1 double mutant accumulated lower levels of trans-zeatin riboside monophosphate (tZRMP) and N(6)-(Delta(2)-isopentenyl)adenosine monophosphate (iPRMP), which are biosynthetic intermediates of bioactive cytokinins. The double mutant also displayed decreased response to exogenous cytokinin in both callus-formation and inhibition-of-hypocotyl-elongation assays. CONCLUSIONS/SIGNIFICANCE: Taken together, our data suggest that in plants AtSOFL1 and AtSOFL2 work redundantly as positive modulators in the fine-tuning of specific cytokinin levels as well as responsiveness.

Enhancement of flowering and branching phenotype in chrysanthemum by expression of ipt under the control of a 0.821 kb fragment of the LEACO1 gene promoter.

The cytokinin biosynthesis gene, isopentenyl transferase (ipt), under the control of an 821 bp fragment of the LEACO1 gene promoter (from Lycopersicon esculentum) was introduced into Dendranthema x grandiflorium 'Iridon' (chrysanthemum). LEACO1(0.821kb)-ipt transgenic lines grown in the vegetative state, exhibited a range of phenotypic changes including increased branching and reduced internode lengths. LEACO1(0.821kb)-ipt transgenic lines grown in the generative state, exhibited increased flower bud count that ranged from 3.8- to 6.7-times the number produced by wild-type plants. Dramatic increases in flower number were associated with a delay of flower bud development and a decrease in flower bud diameter. RT-PCR analysis indicated differences in ipt gene expression between individual transgenic lines that exhibited a range of phenotypes. Within an individual transgenic line, RT-PCR analysis revealed changes in ipt gene expression at different stages of generative shoot development. Expression of ipt in transgenic lines correlated well with high concentrations of the sum total to bioactive cytokinins plus the glucosides and phosphate derivatives of these species, under both vegetative and generative growth conditions. In general, transgenic lines accumulated higher concentrations of both storage-form cytokinins (O-glucosides) and deactivated-form cytokinins (N-glucosides) in generative shoots of than in vegetative shoots. Based on the range of phenotypes observed in various transgenic chrysanthemum lines, we conclude that the LEACO1 (0.821kb) -ipt gene appears to have great potential for use in ornamental crop improvement.

Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux.

Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta. Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.

Senescence progression in a single darkened cotyledon depends on the light status of the other cotyledon in Cucurbita pepo (zucchini) seedlings: potential involvement of cytokinins and cytokinin oxidase/dehydrogenase activity.

Darkness mediates different senescence-related responses depending on the targeting of dark treatment (whole plants or individual leaves) and on the organs that perceive the signal (leaves or cotyledons). As no data are available on the potential role of darkness to promote senescence when applied to individual cotyledons, we have investigated how darkness affects the progression of senescence in either a single or both individually darkened cotyledons of young 10-day-old Cucurbita pepo (zucchini) seedlings. Strong acceleration of senescence was observed when both cotyledons were darkened as judged by the damage in their anatomical structure, deterioration of chloroplast ultrastructure in parallel with decreased photosynthetic rate and photochemical quantum efficiency of PSII. In addition, the endogenous levels of cytokinins (CKs) and IAA were strongly reduced. In a single individually darkened cotyledon, the structure and function of the photosynthetic apparatus as well as the contents of endogenous CKs and IAA were much less affected by darkness, thus suggesting inhibitory effect of the illuminated cotyledon on the senescence of the darkened one. Apparently, the effect of darkness to accelerate/delay senescence in a single darkened cotyledon depends on the light status of the other cotyledon from the pair. The close positive correlation between CK content and the activity of CK oxidase/dehydrogenase (CKX; EC 1.4.3.18/1.5.99.12) suggested that CKX was essentially involved in the mechanisms of downregulation of endogenous CK levels. Our results indicated that CKX-regulated CK signaling could be a possible regulatory mechanism controlling senescence in individually darkened cotyledons.

Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis.

High concentrations of cytokinins (CKs) in the cultivation medium can induce partial photomorphogenesis in dark-grown Arabidopsis seedlings. However, no significant increases in endogenous CK levels have been found in de-etiolated mutants, suggesting that either parallel pathways are involved in the light and CK responses, or changes in the sensitivity to CKs occur during photomorphogenesis. Here it is shown that even modest increases in endogenous CK levels induced by transgenic expression of the CK biosynthetic gene, ipt, can lead to many typical features of light-induced de-etiolation, including inhibition of hypocotyl elongation and partial cotyledon opening. In addition, significant changes in expression of 37 proteins (mostly related to chloroplast biogenesis, a major element of light-induced photomorphogenesis) were detected by image and mass spectrometric analysis of two-dimensionally separated proteins. The identified chloroplast proteins were all up-regulated in response to increased CKs, and more than half are up-regulated at the transcript level during light-induced photomorphogenesis according to previously published transcriptomic data. Four of the up-regulated chloroplast proteins identified here have also been shown to be up-regulated during light-induced photomorphogenesis in previous proteomic analyses. In contrast, all differentially regulated mitochondrial proteins (the second largest group of differentially expressed proteins) were down-regulated. Changes in the levels of several tubulins are consistent with the observed morphological alterations. Further, 10 out of the 37 differentially expressed proteins detected have not been linked to either photomorphogenesis or CK action in light-grown Arabidopsis seedlings in previously published transcriptomic or proteomic analyses.

Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation.

To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays L.) transformants harbouring the ZOG1 gene (encoding a zeatin O-glucosyltransferase from Phaseolus lunatus L.) under the control of the constitutive ubiquitin (Ubi) promoter were generated. The roots and leaves of the transformants had greatly increased levels of zeatin-O-glucoside. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin-deficient plants, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. Transformant leaves had a higher chlorophyll content and increased levels of active cytokinins compared with those of non-transformed sibs. The Ubi:ZOG1 plants exhibited delayed senescence when grown in the spring/summer. While hemizygous transformants had reduced tassels with fewer spikelets and normal viable pollen, homozygotes had very small tassels and feminized tassel florets, resembling tasselseed phenotypes. Such modifications of the reproductive phase were unexpected and demonstrate a link between cytokinins and sex-specific floral development in monocots.

Effects of conditional IPT-dependent cytokinin overproduction on root architecture of Arabidopsis seedlings.

Cytokinin (CK) has been known to inhibit primary root elongation and suggested to act as an auxin antagonist in the regulation of lateral root (LR) formation. While the role of auxin in root development has been thoroughly studied, the detailed and overall description of CK effects on root system morphology, particularly that of developing lateral root primordia (LRPs), and hence its role in organogenesis is still in progress. Here we examine the effects of conditional endogenous CK overproduction on root architecture and consider its temporal aspect during the early development of Arabidopsis thaliana. We employed the pOp/LhGR system to induce ectopic ipt overexpression with a glucocorticoid dexamethasone at designated developmental points. The transient CaMV 35S>GR>ipt transactivation greatly enhanced levels of biologically active CKs of zeatin (Z)-type and identified a distinct developmental interval during which primary root elongation is susceptible to increases in endogenous CK production. Long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem, disturbed a characteristic graded distribution pattern of auxin response in LRPs and impaired their development. Our findings indicate the impact of perturbed endogenous CK on the regulation of asymmetric auxin distribution during LRP development and imply that there is cross-talk between auxin and CK during organogenesis in A. thaliana.

The role of cytokinins in responses to water deficit in tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under 35S or SAG12 promoters.

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans-zeatin O-glucosyltransferase gene (35S::ZOG1), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT. As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves. During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.

Methyl jasmonate is a more effective senescence-promoting factor in Cucurbita pepo (zucchini) cotyledons when compared with darkness at the early stage of senescence.

The effects of short-term darkening and methyl jasmonate (MeJA) on cotyledon senescence were studied 24h after transfer of intact 7-day-old Cucurbita pepo (zucchini) seedlings to darkness or spraying with 100 microM MeJA. The jasmonate inhibitory effect on chlorophyll content and chloroplast transcriptional activity was stronger compared with darkness. Further, MeJA reduced the photosynthetic rate whereas darkness did not affect photosynthesis. Neither stress factor affected the photochemical quantum efficiency of photosystem II (PSII) estimated by the variable fluorescence (F(v))/maximal fluorescence (F(m)) ratio, suggesting the existence of mechanisms protecting the functional activity of PSII at earlier stages of senescence, thus making this parameter more stable compared to others used to quantify senescence. Both stress factors caused a decrease in the content of physiologically active cytokinins, especially trans-zeatin (Z), with the jasmonate effect being much more pronounced when compared to darkness. Our results indicate that MeJA is a more potent inducer of senescence in zucchini cotyledons, at least within the relatively short period of the 24h treatment. This is likely due to its stronger down-regulatory effect on the levels of physiologically active cytokinins.