Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Thermoperiod affects the diurnal cycle of nitrate reductase expression and activity in pineapple plants by modulating the endogenous levels of cytokinins

Nitrate reductase (NR, EC 1.6.6.1) activity in higher plants is regulated by a variety of environmental factors and oscillates with a characteristic diurnal rhythm. In this study, we have demonstrated that the diurnal cycle of NR expression and activity in pineapple (Ananas comosus, cv. Smooth Cayenne) can be strongly modified by changes in the day/night temperature regime. Plants grown under constant temperature (28 degrees C light/dark) showed a marked increase in the shoot NR activity (NRA) during the first half of the light period, whereas under thermoperiodic conditions (28 degrees C light/15 degrees C dark) significant elevations in the NRA were detected only in the root tissues at night. Under both conditions, increases in NR transcript levels occurred synchronically about 4 h prior to the corresponding elevation of the NRA. Diurnal analysis of endogenous cytokinins indicated that transitory increases in the levels of zeatin, zeatin riboside and isopentenyladenine riboside coincided with the accumulation of NR transcripts and preceded the rise of NRA in the shoot during the day and in the root at night, suggesting these hormones as mediators of the temperature-induced modifications of the NR cycle. Moreover, these cytokinins also induced NRA in pineapple when applied exogenously. Altogether, these results provide evidence that thermoperiodism can modify the diurnal cycle of NR expression and activity in pineapple both temporally and spatially, possibly by modulating the day/night changes in the cytokinin levels. A potential relationship between the day/night NR cycle and the photosynthetic pathway performed by the pineapple plants (C(3) or CAM) is also discussed.

Kiwifruit bud release from dormancy: effect of exogenous cytokinins

A new formulate containing citokinins, that is commercialized as Cytokin, has been introduced as dormancy breaking agents. During a three-years study, Cytokin was applied at different concentrations and application times in two producing areas of the Emilia-Romagna region to verify its efficacy as a DBA. Cytokin application increased the bud break and showed a lateral flower thinning effect. Moreover, treated vines showed an earlier and more uniform flowering as compared to control ones. Results obtained on the productive performance revealed a constant positive effect in the fruit fresh weight at harvest. Moreover, Cytokin did not cause any phytotoxicity even at the highest concentrations. Starting from the field observation, which suggested the involvement of cytokinins in kiwifruit bud release from dormancy, 6-BA was applied in open field condition and molecular and histological analyses were carried out in kiwifruit buds collected starting from the endo dormant period up to complete bud break to compare the natural occurring situation to the one induced by exogenous cytokinin application. In details, molecular analyses were set up on to verify the expression of genes involved in the reactivation of cell cycle: cyclin D3, histone H4, cyclin-dependent kinase B, as well as of others which are known to be up regulated during bud release in other species, i.e.isopenteniltransferases (IPTs), which catalyze the first step in the CK biosynthesis, and sucrose synthase 1 and A, which are involved in the sugar supplied. Moreover, histological analyses of the cell division rate in kiwifruit bud apical meristems were performed. These analyses showed a reactivation of the cell divisions during bud release and changes in the expression level of the investigated genes.

Anticancer activity of natural cytokinins: a structure-activity relationship study

Cytokinin ribosides (N(6)-substituted adenosine derivatives) have been shown to have anticancer activity both in vitro and in vivo. This study presents the first systematic analysis of the relationship between the chemical structure of cytokinins and their cytotoxic effects against a panel of human cancer cell lines with diverse histopathological origins. The results confirm the cytotoxic activity of N(6)-isopentenyladenosine, kinetin riboside, and N(6)-benzyladenosine and show that the spectrum of cell lines that are sensitive to these compounds and their tissues of origin are wider than previously reported. The first evidence that the hydroxylated aromatic cytokinins (ortho-, meta-, para-topolin riboside) and the isoprenoid cytokinin cis-zeatin riboside have cytotoxic activities is presented. Most cell lines in the panel showed greatest sensitivity to ortho-topolin riboside (IC(50)=0.5-11.6 microM). Cytokinin nucleotides, some synthesized for the first time in this study, were usually active in a similar concentration range to the corresponding ribosides. However, cytokinin free bases, 2-methylthio derivatives and both O- and N-glucosides showed little or no toxicity. Overall the study shows that structural requirements for cytotoxic activity of cytokinins against human cancer cell lines differ from the requirements for their activity in plant bioassays. The potent anticancer activity of ortho-topolin riboside (GI(50)=0.07-84.60 microM, 1st quartile=0.33 microM, median=0.65 microM, 3rd quartile=1.94 microM) was confirmed using NCI(60), a standard panel of 59 cell lines, originating from nine different tissues. Further, the activity pattern of oTR was distinctly different from those of standard anticancer drugs, suggesting that it has a unique mechanism of activity. In comparison with standard drugs, oTR showed exceptional cytotoxic activity against NCI(60) cell lines with a mutated p53 tumour suppressor gene. oTR also exhibited significant anticancer activity against several tumour models in in vivo hollow fibre assays.

Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation1

This study considered cytokinin distribution in tobacco (Nicotiana tabacum L.) shoot apices in distinct phases of development using immunocytochemistry and quantitative tandem mass spectrometry. In contrast to vegetative apices and flower buds, we detected no free cytokinin bases (zeatin, dihydrozeatin, or isopentenyladenine) in prefloral transition apices. We also observed a 3-fold decrease in the content of cytokinin ribosides (zeatin riboside, dihydrozeatin riboside, and isopentenyladenosine) during this transition phase. The group concluded that organ formation (e.g. leaves and flowers) is characterized by enhanced cytokinin content, in contrast to the very low endogenous cytokinin levels found in prefloral transition apices, which showed no organogenesis. The immunocytochemical analyses revealed a differing intracellular localization of the cytokinin bases. Dihydrozeatin and isopentenyladenine were mainly cytoplasmic and perinuclear, whereas zeatin showed a clear-cut nuclear labeling. To our knowledge, this is the first time that this phenomenon has been reported. Cytokinins do not seem to act as positive effectors in the prefloral transition phase in tobacco shoot apices. Furthermore, the differences in distribution at the cellular level may be indicative of a specific physiological role of zeatin in nuclear processes.

cis-Isomers of Cytokinins Predominate in Chickpea Seeds throughout Their Development1

Trans-isomers of cytokinins (CK) are thought to predominate and have greater biological activity than corresponding cis-isomers in higher plants. However, this study demonstrates a system within which the predominant CK are cis-isomers. CK were measured at four developmental stages in developing chickpea (Cicer arietinum L. cultivar Kaniva) seeds by gas chromatography-mass spectrometry. Concentrations were highest at an early endospermic fluid stage and fell considerably when the cotyledons expanded. The cis-isomers of zeatin nucleotide ([9R-MP]Z), zeatin riboside ([9R]Z), and zeatin (Z) were present in greater concentrations than those of corresponding trans-isomers: (trans)[9R-MP]Z, (trans)[9R]Z, (trans)Z, or dihydrozeatin riboside. Dihydrozeatin, dihydrozeatin nucleotide, and the isopentenyl-type CK concentrations were either low or not detectable. Root xylem exudates also contained predominantly cis-isomers of [9R-MP]Z and [9R]Z. Identities of (cis)[9R]Z and (cis)Z were confirmed by comparison of ion ratios and retention indices, and a full spectrum was obtained for (cis)[9R]Z. Tissues were extracted under conditions that minimized the possibility of RNase hydrolysis of tRNA following tissue disruption, being a significant source of the cis-CK. Since no isomerization of (trans)[2H]CK internal standards occurred, it is unlikely that the cis-CK resulted from enzymic or nonenzymic isomerization during extraction. Although quantities of total CK varied, similar CK profiles were found among three different chickpea cultivars and between adequately watered and water-stressed plants. Developing chickpea seeds will be a useful system for investigating the activity of cis-CK or determining the origin and metabolism of free CK.

Xylem-borne cytokinins: still in search of a role?

Leaf expansion and xylem cytokinin concentration ([X-CK]) decrease in response to nitrogen (N) deprivation. Debate continues over cause, effect, and correlation. Supporting studies provide, at best, correlative evidence that [X-CK] controls leaf growth in response to N-deprivation, while dissenting studies indicate that leaf growth responses to N can be independent of changes in X-CK supply to leaves. A model is proposed to evaluate the physiological significance to leaf growth of changes in plant and environment N concentrations, and plant CK concentrations.

The shoot controls zeatin riboside export from pea roots. Evidence from the branching mutant rms4

The rms4 mutant of pea (Pisum sativum L.) was used in grafting studies and cytokinin analyses of the root xylem sap to provide evidence that, at least for pea, the shoot can modify the import of cytokinins from the root. The rms4 mutation, which confers a phenotype with increased branching in the shoot, causes a very substantial decrease (down to 40-fold less) in the concentration of zeatin riboside (ZR) in the xylem sap of the roots. Results from grafts between wild-type (WT) and rms4 plants indicate that the concentration of cytokinins in the xylem sap of the roots is determined almost entirely by the genotype of the shoot. WT scions normalize the cytokinin concentration in the sap of rms4 mutant roots, whereas mutant scions cause WT roots to behave like those of self-grafted mutant plants. The mechanism whereby rms4 shoots of pea cause a down-regulation in the export of cytokinins from the roots is unknown at this time. However, our data provide evidence that the shoot transmits a signal to the roots and thereby controls processes involved in the regulation of cytokinin biosynthesis in the root.

Rapid increases in cytokinin concentration in lateral buds of chickpea (Cicer arietinum L) during release of apical dominance

We examined the role of cytokinins (CKs) in release of apical dominance in lateral buds of chickpea (Cicer arietinum L.). Shoot decapitation or application of CKs (benzyladenine, zeatin or dihydrozeatin) stimulated rapid bud growth. Time-lapse video recording revealed growth initiation within 2 h of application of 200 pmol benzyladenine or within 3 h of decapitation. Endogenous CK content in buds changed little in the first 2 h after shoot decapitation, but significantly increased by 6 h, somewhat later than the initiation of bud growth. The main elevated CK was zeatin riboside, whose content per bud increased 7-fold by 6 h and 25-fold by 24 h. Lesser changes were found in amounts of zeatin and isopentenyl adenine CKs. We have yet to distinguish whether these CKs are imported from the roots via the xylem stream or are synthesised in situ in the buds, but CKs may be part of an endogenous signal involved in lateral bud growth stimulation following shoot decapitation. To our knowledge, this is the first detailed report of CK levels in buds themselves during release of apical dominance.

The rms1 mutant of pea has elevated indole-3-acetic acid levels and reduced root-sap zeatin riboside content but increased branching controlled by graft-transmissible signal(s)

Rms1 is one of the series of five ramosus loci in pea (Pisum sativum L.) in which recessive mutant alleles confer increased branching at basal and aerial vegetative nodes. Shoots of the nonallelic rms1 and rms2 mutants are phenotypically similar in most respects. However, we found an up to 40-fold difference in root-sap zeatin riboside ([9R]Z) concentration between rms1 and rms2 plants. Compared with wild-type (WT) plants, the concentration of [9R]Z in rms1 root sap was very low and the concentration in rms2 root sap was slightly elevated. To our knowledge, the rms1 mutant is therefore the second ramosus mutant (rms4 being the first) to be characterized with low root-sap [9R]Z content. Like rms2, the apical bud and upper nodes of rms1 plants contain elevated indole-3-acetic acid levels compared with WT shoots. Therefore, the rms1 mutant demonstrates that high shoot auxin levels and low root-sap cytokinin levels are not necessarily correlated with increased apical dominance in pea. A graft-transmissible basis of action has been demonstrated for both mutants from reciprocal grafts between mutant and WT plants. Branching was also largely inhibited in rms1 shoots when grafted to rms2 rootstocks, but was not inhibited in rms2 shoots grafted to rms1 rootstocks. These grafting results are discussed, along with the conclusion that hormone-like signals other than auxin and cytokinin are also involved.

Multiple shoot formation in lentil (Lens culinaris) seeds

A protocol based on seed culture was developed for efficient in vitro propagation of lentil (Lens culinaris Medik). Benzyladenine (BA), thidiazuron (TDZ), and kinetin all induced multiple shoot formation. In terms of the number of long shoots (>2.0 cm) produced per seed, BA and TDZ at optimum concentrations (0.2-0.4 and 0.1 mg/litre, respectively) had similar efficiency, whereas kinetin produced less shoots. Murashige and Skoog (MS) salt composition was better than that of Gamborge (B5) for shoot induction. Increasing calcium (Ca) concentration was necessary to overcome shoot-tip necrosis. For shoot elongation, fresh medium of the same composition of shoot induction medium could be used for stumps from medium with low BA (

Spatial and temporal changes in multiple hormone groups during lateral bud release shortly following apex decapitation of chickpea (Cicer arietinum) seedlings

Although the co-ordination of promotive root-sourced cytokinin (CK) and inhibitory shoot apex-sourced auxin (IAA) is central to all current models on lateral bud dormancy release, control by those hormones alone has appeared inadequate in many studies. Thus it was hypothesized that the IAA : CK model is the central control but that it must be considered within the relevant timeframe leading to lateral bud release and against a backdrop of interactions with other hormone groups. Therefore, IAA and a wide survey of cytokinins (CKs), were examined along with abscisic acid (ABA) and polyamines (PAs) in released buds, tissue surrounding buds and xylem sap at 1 and 4 h after apex removal, when lateral buds of chickpea are known to break dormancy. Three potential lateral bud growth inhibitors, IAA, ABA and cis-zeatin 9-riboside (ZR), declined sharply in the released buds and xylem following decapitation. This is in contrast to potential dormancy breaking CKs like trans-ZR and trans-zeantin 9-riboside 5'phosphate (ZRMP), which represented the strongest correlative changes by increasing 3.5-fold in xylem sap and 22-fold in buds. PAs had not changed significantly in buds or other tissues after 4 h, so they were not directly involved in the breaking of bud dormancy. Results from the xylem and surrounding tissues indicated that bud CK increases resulted from a combination synthesis in the bud and selective loading of CK nucleotides into the xylem from the root.

Cultivo in vitro de Crambe abyssinica Hochst. : germinação, micropropagação, estabilidade genética e anatomia foliar

Crambe (Crambe abyssinica) pertence à família Brassicaceae, originário da Etiópia e principalmente destinado à produção de forragem (30 a 32% de proteína bruta). Atualmente, tem sido bastante cultivado visando à extração de óleo vegetal. Com os atuais incentivos à busca de fontes de energias renováveis, o cultivo de crambe vem ganhando papel de destaque na produção de biodiesel por suas diversas vantagens, como: (a) rápido ciclo de vida (colhida em torno de 90 dias); (b) alta produção de biomassa; (c) alta produtividade de sementes (1000 e 1500 kg ha-1); (d) menor custo de produção em relação a outras fontes oleaginosas, como, canola, girassol e soja; (e) um percentual de óleo total na semente entre 32 e 38%, superando, por exemplo, a soja; (f) potencial de fitorremediação, eficiente na descontaminação de arsênio, cromo e outros metais pesados; e (g) elevado percentual de ácido erúcico (50 a 60%) sendo útil na indústria de plástico e lubrificante. Devido aos poucos trabalhos realizados com crambe, abre-se um vasto campo de investigações científicas que tenham como objetivo desenvolver as potencialidades dessa cultura e, consequentemente, melhorar os aspectos agronômicos e tecnológicos para seu emprego na indústria de biodiesel. Nesse contexto, as técnicas de cultivo in vitro foram importantes tanto para a propagação massal, quanto como ferramenta para uma possível aplicação de outras técnicas biotecnológicas, contribuindo para uma produção homogênea, fiel e em larga escala. Portanto, este trabalho teve como objetivo geral avaliar as condições mais favoráveis à germinação, estabelecimento in vitro e micropropagação de Crambe abyssinica Hochst., além de verificar possíveis alterações genéticas e anatômicas, possibilitando a regeneração e produção de plântulas viáveis. Para a germinação e estabecimento in vitro de crambe, as condições mais favoráveis foram em meio B5 ou WPM, na presença ou ausência de pericarpo e na presença de luz. Na micropropagação dessa espécie, uma frequência satisfatória de regeneração de brotos foi obtida a partir de segmentos apicais utilizados como explante em meio contendo 5 μM de BAP (6- benzilaminopurina), e o alongamento foi satisfatório com 1 μM de GA3 (ácido giberélico). Os marcadores moleculares ISSR (Inter-Simples Sequence Repeats) utilizados para a análise da estabilidade genética indicaram que o segmento apical de crambe é um explante confiável para a micropropagação de plantas geneticamente verdadeiras (true-to-tipe), ou seja, mantém a estabilidade genética. As diversas fontes de citocininas e concentrações utilizadas neste trabalho não promoveram mudanças, no sentido de alterar a organização e/ou a espessura em relação ao controle, e as alterações observadas na estrutura e espessura das folhas dos tratamentos de aclimatização prejudicaram o processo de estabelecimento da plântula ex vitro. Contudo, existe a necessidade de um enraizamento e aclimatização eficiente para completa propagação in vitro de crambe. Portanto, este protocolo de regeneração de plantas in vitro de crambe pode ser útil no processo de criação e desenvolvimento de novas cultivares em um tempo mais curto e no melhoramento genético usando explantes apicais.

Inducción sistémica de la biosintesis de metabolitos secundarios en plantas aromáticas mediada por rizobacterias. Systemic induction of secondary metablolites biosynthesis in aromatic plants by rhizobacteria.

El uso de microorganismos como inoculantes para incrementar la disponibilidad y toma de nutrientes por parte de los cultivos, es una nueva tecnología que ha dado buenos resultados, observándose un incremento en la emergencia, vigor, mayor desarrollo en la parte aérea y de raíces, registrándose aumentos considerables de los rendimientos en cultivos de interés comercial. Esto es debido a que los microorganismos PGPR (Plant Growth promoting rhizobacteria) sintetizan ciertas sustancias reguladoras del crecimiento como giberelinas, citoquininas y auxinas; las cuales estimulan la densidad y longitud de los pelos radicales, aumentando así la cantidad y longitud de las raíces de los vegetales. Así, se incrementa la capacidad de absorción de agua y nutrientes, haciendo que las plantas sean más vigorosas, productivas y tolerantes a condiciones climáticas adversas, como sequías o heladas. Otro factor benéfico es que ciertos microorganismos solubilizan nutrientes poco móviles en el suelo como el caso del fósforo, segundo nutriente, después del nitrógeno en importancia para el crecimiento de los cultivos. Estos microorganismos también tienen una función muy importante en el control natural de agentes patógenos, a través de la inducción del sistema de defensa en las plantas, aumentando su resistencia a enfermedades, a través de la producción de compuestos bacterianos como antibióticos y sideróforos. Los variados mecanismos mediante los cuales la acción PGPR se lleva a cabo no son plenamente conocidos y, por lo tanto, es necesario determinar con precisión su efecto particular en la biología de la planta beneficiada. Las plantas aromáticas y medicinales inoculadas con microorganismos (rizobacterias) registran un incremento en varios parámetros de crecimiento vegetal (peso fresco parte aérea, peso seco de raíz, número de hojas, etc) y en el rendimiento de aceite esencial (AE). El aumento de la síntesis, y la variación de los porcentajes relativos de los componentes principales de AE en plantas aromáticas, como efecto de la inoculación, podría considerarse como una respuesta defensiva de la planta frente a la colonización de microorganismos dado que varios AE poseen propiedades antimicrobianas. El incremento de estos metabolitos también se ha registrado como respuesta frente a la herbivoría. En el presente proyecto se propone dilucidar la existencia de una relación entre las defensas inducidas por rizobacterias con la producción de metabolitos secundarios en plantas aromaticas y medicinales. The use of microorganisms as inoculants to increase the availability and nutrient uptake by crops, is a new technology that has been successfully applied, with an increase in the emergence, vigor, greater development in the shoot and roots, recording significant increases in yields of crop with commercial interest. This is because microorganisms PGPR (Plant Growth Promoting rhizobacteria) synthesize certain growth regulating substances such as gibberellins, cytokinins and auxins, which stimulate the density and length of root hairs, increasing the number and length of roots. Thus, increase the capacity of absorbing water and nutrients, make the plants more vigorous, productive and tolerant to adverse climatic conditions such as drought or frost.Another beneficial factor is that some microorganisms solubilize nutrients mobile in the soil as the case of phosphorus, second nutrient after nitrogen important for plant growth. These organisms also have an important role in the natural control of pathogens through the induction of the plants defense system, increasing their resistance to disease through the production of compounds such as antibiotics and bacterial siderophores. The various mechanisms by which PGPR action takes place are not fully known and therefore it is necessary to accurately determine its particular effect on the biology of the specific plant benefit. Aromatic and medicinal plants inoculated with microorganisms (rhizobacteria) recorded an increase in several parameters of plant growth (shoot fresh weight, root dry weight, leaf number, etc) and essential oil yield (AE). The increase in the biosynthesis, and changes in the relative percentages of the main components of AE in aromatic plants inoculated with rizobacterias, could be regarded as a plant defense response against microbial colonization, since several AE have antimicrobial properties. The increase of these metabolites have also been recorded as a response to herbivory.