Effect Of Cytokinins Research Articles

Effects of β-lactam antibiotics, auxins, and cytokinins on shoot regeneration from callus cultures of two hybrid aspens, Populus tremuloides × P. tremula and P. x canescens × P. gradidentata

The effects of β-lactam antibiotics (penicillin, carbenicillin and cefotaxime), cytokinins, and auxins including phenylacetic acid, a β-lactam breakdown product, were evaluated during in vitro shoot morphogenesis in two hybrid aspens; P. tremuloides × P. tremula (XTTa) and P. x canescens × P. grandidentata (XCaG). Although different callus and shoot induction media were used for both hybrids, the β-lactams often engendered similar responses. At concentrations of 1,000 mg l−1, carbenicillin adversely impacted shoot elongation and, to a lesser degree, shoot regeneration. Cefotaxime enhanced caulogenesis for all of the concentrations evaluated (125–500 mg l−1) especially when the cytokinin thidiazuron was used for shoot induction. The shoots formed faster and in greater numbers; and the improvements were significant (α = 0.05) for both hybrids. However, hyperhydricity was more problematic when cefotaxime was included in the media. The incidence of shoot hyperhydricity for the XCaG hybrid was more than twice as great for the highest cefotaxime concentration evaluated (500 mg l−1) than for the control (>90% vs. ~40%). Penicillin had an opposite effect. Hyperhydricity frequencies for the XCaG hybrid were lower when the media were supplemented with penicllin and the reductions were statistically significant at concentrations of 500–1,000 mg l−1. The effects of the antibiotics were generally not reproduced by the auxins (0.1–100 μM), including phenylacetic acid, or the other potential β-lactam degradation products evaluated (e.g. phenylmalonic acid, aminopenicillanic acid). The antibiotics may have affected shoot hyperhydicity indirectly via changes in the time course of shoot regeneration.

Studies on Seed Germination, Seedling Growth, and In Vitro Shoot Induction of Aloe ferox Mill., a Commercially Important Species

A study was done to investigate the effects of some physical and chemical factors on growth and development of Aloe ferox ex vitro and in vitro. The effects of light, temperature, and smoke–water on seed germination, ex vitro seedling growth requirements, and effect of germination medium and cytokinins on shoot induction and multiplication in vitro were investigated. The highest germination percentage of A. ferox seeds was recorded between 15 and 30 °C. Seed germination was inhibited at 10 and 35 °C. There was a nonsignificant increase in germination percentage (78%) for seeds that were germinated under constant dark conditions. Smoke–water-treated seeds showed significant improvement in percentage germination (80%) over the control (66%) at 25 °C with a 16-h photoperiod. Seedlings of A. ferox subjected to alternating temperatures (30/15 °C) or being irrigated three times weekly at 25 °C showed better seedling growth. In an in vitro experiment, seedlings germinated in distilled water and one-tenth strength Murashige and Skoog medium had superior shoot induction competence compared with the other germination media. The cytokinins meta-Topolin (mT) and meta-Topolin riboside (mTR) at 5 μm gave significantly higher shoot multiplication rates compared with the control and benzyladenine (BA)-treated plants. A higher abnormality index was recorded for BA-treated plants. The findings of this study will be beneficial for commercial propagation of Aloe ferox.

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (Mesembryanthemum crystallinum L.)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO3, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO3 applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.

The cytokinin type-B response regulator PtRR13 is a negative regulator of adventitious root development in Populus.

Adventitious root formation at the base of plant cuttings is an innate de novo organogenesis process that allows massive vegetative propagation of many economically and ecologically important species. The early molecular events following shoot excision are not well understood. Using whole-genome microarrays, we detected significant transcriptome remodeling during 48 h following shoot removal in Populus tremula x Populus alba softwood cuttings in the absence of exogenous auxin, with 27% and 36% of the gene models showing differential abundance between 0 and 6 h and between 6 and 24 h, respectively. During these two time intervals, gene networks involved in protein turnover, protein phosphorylation, molecular transport, and translation were among the most significantly regulated. Transgenic lines expressing a constitutively active form of the Populus type-B cytokinin response regulator PtRR13 (DeltaDDKPtRR13) have a delayed rooting phenotype and cause misregulation of CONTINUOUS VASCULAR RING1, a negative regulator of vascularization; PLEIOTROPIC DRUG RESISTANCE TRANSPORTER9, an auxin efflux transporter; and two APETALA2/ETHYLENE RESPONSE FACTOR genes with sequence similarity to TINY. Inappropriate cytokinin action via DeltaDDKPtRR13 expression appeared to disrupt adventitious root development 24 h after shoot excision, when root founder cells are hypothesized to be sensitive to the negative effects of cytokinin. Our results are consistent with PtRR13 acting downstream of cytokinin to repress adventitious root formation in intact plants, and that reduced cytokinin signaling after shoot excision enables coordinated expression of ethylene, auxin, and vascularization pathways leading to adventitious root development.

Nebularine Affects Plant Growth and Development but does not Interfere with Cytokinin Signaling

Nebularine is known for its high cytotoxicity in animals, whereas in plants it was originally believed to be an anticytokinin. In this study we show that in classical cytokinin bioassays, nebularine antagonized cytokinin function in senescence and callus biotests but not in the Amaranthus bioassay. Nebularine reversed the inhibitory effect of cytokinin on lateral root formation in Arabidopsis seedlings, and when applied alone caused increased lateral root formation and shortening of the main root. Systematic spraying of Arabidopsis plants with nebularine led to yellowing and formation of purple pigments, local drying, and withering, although younger plants showed a greater resilience. Comparison of nebularine cytotoxicity in plant and animal cells showed that the growth of tobacco BY-2 cells was inhibited with only about tenfold lower efficacy than mammalian cell lines. Most importantly, direct binding assay with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 showed that nebularine did not compete for binding with the natural cytokinin trans-zeatin. Although nebularine reduced cytokinin-induced expression of the cytokinin reporter ARR5:GUS in planta, the same effect was observed for DR5:GUS, an auxin reporter gene. Taken together, the results indicate that the mode of action of nebularine does not involve cytokinin signaling and that the anticytokinin-like effect is rather a consequence of the inhibition of various processes as described for animal systems.

Cytokinin regulates root meristem activity via modulation of the polar auxin transport

Plant development is governed by signaling molecules called phytohormones. Typically, in certain developmental processes more than 1 hormone is implicated and, thus, coordination of their overlapping activities is crucial for correct plant development. However, molecular mechanisms underlying the hormonal crosstalk are only poorly understood. Multiple hormones including cytokinin and auxin have been implicated in the regulation of root development. Here we dissect the roles of cytokinin in modulating growth of the primary root. We show that cytokinin effect on root elongation occurs through ethylene signaling whereas cytokinin effect on the root meristem size involves ethylene-independent modulation of transport-dependent asymmetric auxin distribution. Exogenous or endogenous modification of cytokinin levels and cytokinin signaling lead to specific changes in transcription of several auxin efflux carrier genes from the PIN family having a direct impact on auxin efflux from cultured cells and on auxin distribution in the root apex. We propose a novel model for cytokinin action in regulating root growth: Cytokinin influences cell-to-cell auxin transport by modification of expression of several auxin transport components and thus modulates auxin distribution important for regulation of activity and size of the root meristem.

Transgenic grapefruit plants expressing the PAPETALA3-IPT gp gene exhibit altered expression of PR genes

Transgenic grapefruit plants (Citrus paradisi cv. ‘Duncan’) with the isopentenyltransferase (ipt) gene under the control of APETALA3 promoter have been produced using Agrobacterium-mediated transformation. The relative expression level of the ipt gene was between 2.3 and 7 times higher in transformed plants than in the wild-type but despite the presence of a tissue-specific promoter, the expression was not limited only to flower tissue. Increased levels of trans-zeatin riboside between 9.4 and 32-fold found in transgenic grapefruit were considered the consequence of ectopic expression of the ipt gene. Chlorophyll levels in fully expanded uppermost leaves were also about 30% higher in transgenic than in wild-type plants. Involvement of cytokinins in control of expression of three pathogenesis-related protein genes: β-1,3-glucanase, a stress related PR gene 24P220, and an acidic chitinase, 24P262 was examined. Expression of β-1,3-glucanase, and 24P220 gene were significantly enhanced in transgenic plants while the expression of chitinase was reduced to low levels. Our results confirm the effect of cytokinins on expression of genes implicated in the response of grapefruit plants to pathogen attack and suggest a possible role of cytokinins in pathogen resistance.

Role of polarity in de novo shoot bud initiation from stem disc explants of Curculigo orchioides Gaertn. and its encapsulation and storability

The occurrence of strong polarity towards shoot bud induction and the effect of cytokinin(s) on each segment of stem axis, encapsulation and storability of de novo Shoot buds of Curculigo orchioides Gaertn. (Hypoxidaceae) have been documented in the present communication. Maximum number of shoot buds arising de novo from the stem discs (cross section) explanted from proximal end on MS medium fortified with BAP and KIN 1 mg/L each. Stem discs from distal end were less efficient in shoot bud induction. A combination of two cytokinins (BAP and KIN) as a synergistic effect on shoot buds induction from each segment of stem axis. Stem discs in inverted position produced shoot buds from the lower surface, showing strong polarity within the explant. Further, storability and shoot development of sodium alginate encapsulated shoot buds of Curculigo orchioides were tested on half-strength Murashige and Skoog (MS) basal medium fortified with coconut water (10% v/v). The frequency of regeneration from encapsulated shoot buds was affected significantly by concentration of sodium alginate and the duration of exposure to calcium chloride. Shoot buds encapsulated with 2.5% sodium alginate dissolved in MS basal salts solution recorded significantly higher shoot development than other treatments. A relatively short (5 min) incubation with calcium chloride solution provided uniform encapsulation of shoot buds that gave the highest percentage (68%) of shoot development. Encapsulated shoot buds could be stored at 4°C for 50 days without reduction in viability as oppose to non-encapsulated shoot buds, which showed 9.5% viability after 20 days at 4°C. Encapsulated shoot bud developed into normal shoots. Based on the present observations an improved protocol may be developed for the rapid multiplication and conservation of the endangered species—C. orchioides.

Effects of light and cytokinin on in vitro micropropagation and microrhizome production in turmeric (Curcuma longa L.)

Using clonal in vitro propagated plantlets of Curcuma longa, the effects of light and cytokinin on micropropagation and microrhizome formation were investigated. The results showed the promotive effects of low light intensity for micropropagation and of short day condition for well-developed microrhizome induction. In the tissue of well-developed microrhizomes, differentiation of yellowish-pigmented cells similar to the secretory cells of rhizome of the mother plant was observed. Staining of the cells with nile red suggested the accumulation of essential oil. Rapidly growing callus was isolated from the root tip of micropropagated plantlets which retained totipotency during subculture. High concentration of kinetin induced microrhizome formation in this callus. Yellow pigmented cells were also observed in the tissue of microrhizome, although far less frequency.

56. Effects of auxin and cytokinin on strigolactone production and exudation

56. Effects of auxin and cytokinin on strigolactone production and exudation

Effect of cell size and cytokinins on growth of petunia plants

Effect of cell size and cytokinins on growth of petunia plants

Low Levels of Gibberellic Acid Control the Biosynthesis of Ajmalicine inCatharanthus roseusCell Suspension Cultures

In periwinkle cell suspensions, amounts of gibberellic acid ranging from 10 ( - 10) M to 10 ( - 7) M significantly antagonized, in a dose-dependant manner, the stimulation of ajmalicine biosynthesis by cytokinins (CKs). This inhibitory effect was strictly correlated with the abolition of the expression of two genes encoding enzymes of the monoterpenoid indole alkaloid (MIA) biosynthetic pathway and was normally upregulated after CK treatments. Moreover, low concentrations of the gibberellin biosynthesis inhibitor paclobutrazol could reverse the inhibitory effects of low auxin levels on ajmalicine accumulation in the cells. On the other hand, gibberellic acid could not affect the expression of two type-A response regulators considered to be CK primary response genes in periwinkle cells. The antagonistic effects of gibberellins and cytokinins on MIA biosynthesis and their possible impact on elements of the signal transduction are discussed.

The effect of cytokinins on growth and sexual organ development in the gametophyte of Blechnum spicant L.

In this study, we report the role of exogenous and endogenous cytokinins on growth and sexual organ development in the fern Blechnum spicant L. Spore-derived gametophytes (SG) were cultured in full-strength Murashige and Skoog (1962) liquid medium supplemented with (a) 4.44 μMN6-benzyladenine (BAP), (b) a crude extract from mature female gametophytes, and (c) 4.44 μM BAP in combination with the crude extract from mature gametophytes, respectively. Both BAP and the crude extract delayed the gametophyte development, and this effect was increased when they were added together. With respect to sexual organ development, BAP inhibited the sexual organ formation, while the crude extract favored antheridia formation; however, when added together, the percentage of antheridia decreased. The endogenous level of the cytokinins cis-zeatin (cZ), cis-zeatin-riboside (cZR), dihydrozeatin (DHZ), dihydrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), isopentenyl-9-glucoside (iP9G), trans-zeatin (tZ), and trans-zeatin riboside (tZR) were analyzed in female and male gametophytes of B. spicant L. The endogenous levels of cytokinins tZ, cZ, DHZ, cZR, iP, and iPR were higher in female gametophytes than in male gametophytes, with the endogenous iP and iPR content being increased more than 300 and 400 times, respectively.

The effect of cytokinins on the concentration of hydroxyl radicals and the intensity of lipid peroxidation in nitrogen deficient wheat

Senescence of plant tissues is a natural process that may be accelerated by unfavourable environmental factors, including disbalanced mineral nutrition. During plant senescence, the concentration of reactive oxygen species (ROS) increases in the cells, whereas the concentration of cytokinins declines. Negative correlation between ROS, such as hydroxyl radical ( · OH), and concentration of cytokinins triggers the question whether the addition of cytokinins can reduce ROS production and/or its concentration. This would also reduce the level of lipid peroxidation (LP) in the senescing tissue, which is the final phase in the destruction of cell membranes by ROS.With the aim to address these questions, the effects of cytokinins (trans-zeatin, TZ, and benzyladenine, BA) on the concentration of · OH and LP were studied in wheat subjected to nitrogen deficiency. Foliar application of TZ reduced the concentration of · OH and the intensity of LP in the leaves of young wheat plants insufficiently supplied with nitrogen.

In vitro propagation of Achyranthes aspera: Effects of cytokinins and auxins

In vitro propagation of Achyranthes aspera: Effects of cytokinins and auxins

An autophagy-associated Atg8 protein is involved in the responses of Arabidopsis seedlings to hormonal controls and abiotic stresses

Eukaryotes contain a ubiquitous family of autophagy-associated Atg8 proteins. In animal cells, these proteins have multiple functions associated with growth, cancer, and degenerative diseases, but their functions in plants are still largely unknown. To search for novel functions of Atg8 in plants, the present report tested the effect of expression of a recombinant AtAtg8 protein, fused at its N-terminus to green fluorescent protein (GFP) and at its C-terminus to the haemagglutinin epitope tag, on the response of Arabidopsis thaliana plants to the hormones cytokinin and auxin as well as to salt and osmotic stresses. Expression of this AtAtg8 fusion protein modulates the effect of cytokinin on root architecture. Moreover, expression of this fusion protein also reduces shoot anthocyanin accumulation in response to cytokinin feeding to the roots, implying the participation of AtAtg8 in cytokinin-regulated root–shoot communication. External application of cytokinin leads to the formation of novel GFP–AtAtg8-containing structures in cells located in the vicinity of the root vascular system, which are clearly distinct in size and dynamic movement from the GFP–AtAtg8-containing autophagosome-resembling structures that were observed in root epidermis cells. Expression of the AtAtg8 fusion construct also renders the plants more sensitive to a mild salt stress and to a lesser extent to a mild osmotic stress. This sensitivity is also associated with various changes in the root architecture, which are morphologically distinct from those observed in response to cytokinin. The results imply multiple functions for AtAtg8 in different root tissues that may also be regulated by different mechanisms.

Cytokinin Stimulates Chloroplast Transcription in Detached Barley Leaves

Chloroplasts are among the main targets of cytokinin action in the plant cell. We report here on the activation of transcription by cytokinin as detected by run-on assays with chloroplasts isolated from apical parts of first leaves detached from 9-d-old barley (Hordeum vulgare) seedlings and incubated for 3 h on a 2.2 x 10(-5) m solution of benzyladenine (BA). Northern-blot analysis also detected a BA-induced increase in the accumulation of chloroplast mRNAs. A prerequisite for BA activation of chloroplast transcription was preincubation of leaves for 24 h on water in the light, resulting in a decreased chloroplast transcription and a drastic accumulation of abscisic acid. Cytokinin enhanced the transcription of several chloroplast genes above the initial level measured before BA treatment, and in the case of rrn16 and petD even before preincubation. Cytokinin effects on basal (youngest), middle, and apical (oldest) segments of primary leaves detached from plants of different ages revealed an age dependence of chloroplast gene response to BA. BA-induced stimulation of transcription of rrn16, rrn23, rps4, rps16, rbcL, atpB, and ndhC required light during the period of preincubation and was further enhanced by light during the incubation on BA, whereas activation of transcription of trnEY, rps14, rpl16, matK, petD, and petLG depended on light during both periods. Our data reveal positive and differential effects of cytokinin on the transcription of chloroplast genes that were dependent on light and on the age (developmental stage) of cells and leaves.

Differential effects of methyl jasmonate on growth and division of etiolated zucchini cotyledons

The jasmonates are well studied in the context of plant defence but increasingly are also recognised as playing roles in development. In many systems, jasmonates antagonise the effects of cytokinins. The aim of the present work was to elucidate interactions between methyl jasmonate and cytokinin (benzyladenine) in regulating growth of zucchini (Cucurbita pepo L., cv. Cocozelle, var. Tripolis) cotyledons, taking advantage of the ability to simultaneously quantify cell enlargement and division from paradermal sections of the first palisade layer. Growth regulators were applied to cotyledons, excised from dry seeds and grown in darkness. Cytokinin stimulated expansion and division whereas, surprisingly, jasmonate stimulated expansion but inhibited division. Jasmonate antagonised the stimulating effect of cytokinin on division but worked cooperatively with cytokinin in increasing expansion. However, expansion with jasmonate was more isotropic than with cytokinin. Jasmonate also stimulated the loss of cellular inclusions and soluble protein. Soluble proteins revealed a partial antagonism between jasmonate and cytokinin. These results illustrate the complex interplay between jasmonates and cytokinin in the regulatory network of cotyledon development following germination.

Effects of cytokinin and senescence-inducing factors on expression of P ARR5 -GUS gene construct during leaf senescence in transgenic Arabidopsis thaliana plants

ARR5-gene expression was studied in the course of natural leaf senescence and detached leaf senescence in the dark using Arabidopsis thaliana plants transformed with the PARR5-GUS gene construct. GUS-activity was measured as a marker of ARR5-gene expression. Chlorophyll and total protein amounts were also estimated to evaluate leaf senescence. Natural leaf senescence was accompanied by the progressive decline in the GUS-activity in leaves of the 2nd and 3rd nodes studied, and this shift of GUS-activity was more pronounced than the loss of chlorophyll content. The ability of the ARR5-gene promoter to respond to cytokinin was not eliminated during natural leaf senescence, as was demonstrated by a cytokinin-induced increase in GUS activity in leaves after their detachment and incubation on benzyladenine (BA, 5 × 10−6 M) in the dark. Leaf senescence in the dark was associated with the further decrease in the GUS-activity. The ARR5-gene promoter response to cytokinin was enhanced with the increase of the age of plants, taken as a source of leaves for cytokinin treatments. Hence, although the expression of the ARR5 gene reduces during natural and dark/detached leaf senescence, the ARR5-gene sensitivity to cytokinin was maintained in both cases and even increased with the leaf age. This data suggest that the ARR5 gene, which belongs to the type-A negative regulators of plant response to cytokinin, could be a feedback regulator able to prevent retardation by cytokinin of leaf senescence when it is important for plant life. Growth regulators either reduced ARR5 gene response to cytokinin during senescence of mature detached leaves in the dark (SA, meJA, ABA, SP) or increased it (IAA), thus modifying the resulting rate of its expression.

The Inhibitory Effects of Auxin and Cytokinin on Dark- and ABA-induced Stomatal Closure in Broad Bean

In the present studies, the effects and mechanisms of natural and synthetic auxin IAA, NAA, 2,4-D, cytokinin ZT, KT, 6-BA on dark- and ABA-induced stomatal closure were investigated by means of stomatal bioassay and using laser-scanning con- focal microscopy. Isolated epidermal strips of Vicia faba were incubated with IAA (10 μmol L-1), NAA (10 μmol L-1), 2,4-D (10 μmol L-1), ZT (0.1 μmol L-1), KT (0.2 μmol L-1), 6-BA (0.2 μmol L-1), NO scavenger c-PTIO (200 μmol L-1), Hb (100 μmol L-1), NOS inhibitor L-NAME (25 μmol L-1), H2O2 scavenger AsA (100 μmol L-1), CAT (100 U mL-1), inhibitor of H2O2-generating enzyme NADPH oxidase DPI (10 μmol L-1) for 3 h, in darkness or in light in the presence of ABA (1 μmol L-1), respectively. The results showed that auxin, cytokinin, as well as c-PTIO, Hb, L-NAME, AsA, CAT, and DPI, reversed dark- and ABA-induced stomatal closure significantly. Epidermal strips treated with auxin and cytokinin were loaded with NO-fluorescent dye DAF-2DA or H2O2-fluorescent dye H2DCF-DA. The results indicated that darkness and ABA could induce an intense DAF-2DA or H2DCF-DA fluorescence in guard cells. However, dark- and ABA-induced DAF-2DA and H2DCF-DA fluorescence were largely prevented by auxin and cytokinin tested. Similarly, the treatments of c-PTIO, Hb, L-NAME and AsA, CAT, DPI also substantially suppressed dark- and ABA-induced DAF-2DA and H2DCF-DA fluorescence, respectively. These results provide the evidence that auxin and cytokinin tested lessen assuredly NO and H2O2 levels induced by dark and ABA in guard cells. Consider- ing synthetic auxin, cytokinin NAA, 2,4-D, KT, 6-BA and natural IAA, ZT were used in the present work and IAA, ZT are repre- sentative of endogenous auxin and cytokinin respectively, the effects of auxin, cytokinin tested on dark- and ABA-induced stomatal closure and NO, H2O2 level can be attributed to an universal effect of auxin or cytokinin.