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De La Cruz Chacón, Yngrid - One of the best experts on this subject based on the ideXlab platform.
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Efecto de citoquininas en el cultivo in vitro de dos especies de Berries nativos del Perú: Vaccinium floribundum Kunth y Macleanía rupestris Kunth A. C. Smith
'Baishideng Publishing Group Inc.', 2020Co-Authors: De La Cruz Chacón, YngridAbstract:Universidad Nacional Agraria La Molina. Facultad de Ciencias. Departamento Académico de BiologíaLos berries nativos del Peru poseen un elevado valor nutritivo; sin embargo, solo algunas han sido domesticadas. Presentan caracteres muy variables como frutos de diferentes tamanos, calidad y sabor que hacen de estas especies un banco genetico muy importante para los programas de mejoramiento. El objetivo del presente trabajo es evaluar el crecimiento in vitro de dos especies de berries nativos del Peru como Vaccinium floribundum Kunth y Macleania rupestris Kunth A. C. Smith, conocidas como gpushgay h y galicon h, respectivamente; utilizando diferentes medios de cultivos, suplementado con diferentes concentraciones de citoquininas (Zeatina y kinetina). El medio de cultivo basal utilizado fue el medio de Lloyd y McCown (1980), conocido como Woody Plant (WP) y como reguladores de crecimiento, soluciones madre de Zeatina 1000 ppm y kinetina 1000 ppm. Se trabajo en un ambiente controlado de crecimiento, con temperatura de 22 ‹C +/- 2 ‹C, fotoperiodo de 8/16 horas y luminosidad de 47,74 ƒÊmol.m-2.s-1. El diseno experimental fue el completamente al azar (DCA) con cuatro tratamientos y siete repeticiones, durante un periodo de 120 dias. Las variables respuestas fueron longitud del explante, numero de hojas, numero de brotes y numero de raices. Los datos obtenidos fueron analizados por analisis de varianza simple (ANOVA) y la prueba de Tukey, donde se obtuvo diferencias significativas en cada una de las variables respecto a los tratamientos aplicados. Se observo que las caracteristicas fenotipicas fueron mejores en los tratamientos suplementados con kinetina, obteniendose explantes con mayor longitud, entrenudos mas largos, con hojas mas verdes y presencia de raiz; a diferencia con la Zeatina, en donde solo se observaron callos. Se concluyo que el mejor medio de propagacion para gpushgay h es el medio suplementado con kinetina 1.0 ppm y para galicon h es el medio suplementado con kinetina 0.5 ppm.Peruvian native berries have a high nutritional value; however, only few of them have been domesticated. Their fruits have different sizes, quality and flavor that make these species a very important genetic bank for breeding programs. The objective of this research is to assess the in vitro propagation of two species of Peruvian native berries, namely, Vaccinium floribundum Kunth and Macleania rupestris Kunth A. C. Smith -known as “pushgay” and “alicon”, respectively- using different culture media, supplemented with different cytokinin concentrations (Zeatin and kinetin). The basal culture medium used was Lloyd and McCown´s medium (1980), known as Woody Plant (WP), and, as growth regulators, stock solutions of Zeatin 1000 ppm or kinetin 1000 ppm. Work was carried out in a controlled environment, with a temperature of 22 ºC +/- 2 ºC, photoperiod of 8/16 hours and 47.74 μmol.m-2.s-1 luminosity. The experimental design used was a completely random (DCA), involving four treatments and seven repetitions, during a period of 120 days. The response variables were length of the explant, number of leaves, number of shoots and number of roots. The data were analyzed by a simple variance analysis (ANOVA) and Tukey test, where significant differences were obtained in each of the variables. It was observed that the treatments supplement with kinetin were better, resulting in explants with longer length, longer internodes, greener leaves and root presence, whereas in the treatment with Zeatin, only calluses were observed. It was concluded that the propagation medium for “pushgay” is the one supplemented with 1.0 ppm kinetin and, for “alicon” the medium supplemented with 0.5 ppm kinetin
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Efecto de citoquininas en el cultivo in vitro de dos especies de Berries nativos del Perú: Vaccinium floribundum Kunth y Macleanía rupestris Kunth A. C. Smith
'Universidad Nacional Agraria la Molina', 2020Co-Authors: De La Cruz Chacón, YngridAbstract:Universidad Nacional Agraria La Molina. Facultad de Ciencias. Departamento Académico de BiologíaLos berries nativos del Peru poseen un elevado valor nutritivo; sin embargo, solo algunas han sido domesticadas. Presentan caracteres muy variables como frutos de diferentes tamanos, calidad y sabor que hacen de estas especies un banco genetico muy importante para los programas de mejoramiento. El objetivo del presente trabajo es evaluar el crecimiento in vitro de dos especies de berries nativos del Peru como Vaccinium floribundum Kunth y Macleania rupestris Kunth A. C. Smith, conocidas como gpushgay h y galicon h, respectivamente; utilizando diferentes medios de cultivos, suplementado con diferentes concentraciones de citoquininas (Zeatina y kinetina). El medio de cultivo basal utilizado fue el medio de Lloyd y McCown (1980), conocido como Woody Plant (WP) y como reguladores de crecimiento, soluciones madre de Zeatina 1000 ppm y kinetina 1000 ppm. Se trabajo en un ambiente controlado de crecimiento, con temperatura de 22 ‹C +/- 2 ‹C, fotoperiodo de 8/16 horas y luminosidad de 47,74 ƒÊmol.m-2.s-1. El diseno experimental fue el completamente al azar (DCA) con cuatro tratamientos y siete repeticiones, durante un periodo de 120 dias. Las variables respuestas fueron longitud del explante, numero de hojas, numero de brotes y numero de raices. Los datos obtenidos fueron analizados por analisis de varianza simple (ANOVA) y la prueba de Tukey, donde se obtuvo diferencias significativas en cada una de las variables respecto a los tratamientos aplicados. Se observo que las caracteristicas fenotipicas fueron mejores en los tratamientos suplementados con kinetina, obteniendose explantes con mayor longitud, entrenudos mas largos, con hojas mas verdes y presencia de raiz; a diferencia con la Zeatina, en donde solo se observaron callos. Se concluyo que el mejor medio de propagacion para gpushgay h es el medio suplementado con kinetina 1.0 ppm y para galicon h es el medio suplementado con kinetina 0.5 ppm.Peruvian native berries have a high nutritional value; however, only few of them have been domesticated. Their fruits have different sizes, quality and flavor that make these species a very important genetic bank for breeding programs. The objective of this research is to assess the in vitro propagation of two species of Peruvian native berries, namely, Vaccinium floribundum Kunth and Macleania rupestris Kunth A. C. Smith -known as “pushgay” and “alicon”, respectively- using different culture media, supplemented with different cytokinin concentrations (Zeatin and kinetin). The basal culture medium used was Lloyd and McCown´s medium (1980), known as Woody Plant (WP), and, as growth regulators, stock solutions of Zeatin 1000 ppm or kinetin 1000 ppm. Work was carried out in a controlled environment, with a temperature of 22 ºC +/- 2 ºC, photoperiod of 8/16 hours and 47.74 μmol.m-2.s-1 luminosity. The experimental design used was a completely random (DCA), involving four treatments and seven repetitions, during a period of 120 days. The response variables were length of the explant, number of leaves, number of shoots and number of roots. The data were analyzed by a simple variance analysis (ANOVA) and Tukey test, where significant differences were obtained in each of the variables. It was observed that the treatments supplement with kinetin were better, resulting in explants with longer length, longer internodes, greener leaves and root presence, whereas in the treatment with Zeatin, only calluses were observed. It was concluded that the propagation medium for “pushgay” is the one supplemented with 1.0 ppm kinetin and, for “alicon” the medium supplemented with 0.5 ppm kinetin.Tesi
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Multiplicación in vitro de Macleania rupestris (Kunth) A.C. Sm. (Ericaceae)
Instituto de Biotecnología de las Plantas, 2019Co-Authors: Gutiérrez Rosati, Antonietta Ornella, De La Cruz Chacón, YngridAbstract:Macleania rupestris (Kunth) A.C. Smith (Ericaceae) is a plant native to the Andes, which produces edible fruits with great food and commercial potential. M. rupestris plants have been in vitro established from seeds of ripe fruits and to increase their number it is necessary to define the conditions of their multiplication. The objective of the investigation was to determine the effect of Zeatin and kinetin to in vitro multiply M. rupestris plants. As a basal culture medium, Woody Plant (WP) was used. Treatments included the CIRGEBB culture medium (WP + 0.5 mg l-1 Zeatin), A (WP + 0.5 mg l-1 kinetin), B (WP + 1.0 mg l-1 kinetin) and WP as control. The variables evaluated were explant height (cm), number of shoots and number of leaves, at 60 and 120 days of culture. The use of Zeatin and kinetin in the WP culture medium allow in vitro multiplication of M. rupestris. However, the addition of Zeatin favors the development of plants with phenotypic characteristics suitable for micropropagation. Based on the results, it is proposed to use the culture medium A (WP + 0.5 mg l-1 kinetin).Macleania rupestris (Kunth) A.C. Smith (Ericaceae) es una planta originaria de los Andes, que produce frutos comestibles con gran potencial alimenticio y comercial. Se han establecido in vitro plantas de M. rupestris a partir de semillas de frutos maduros y para incrementar su número se requiere definir las condiciones de su multiplicación. El objetivo de la investigación fue determinar el efecto de Zeatina y kinetina para multiplicar in vitro plantas de M. rupestris. Como medio de cultivo basal se utilizó Woody Plant (WP). Los tratamientos incluyeron el medio de cultivo CIRGEBB (WP+Zeatina 0.5 mg l-1), A (WP+kinetina 0.5 mg l-1), B (WP+kinetina 1.0 mg l-1) y WP como control. Las variables evaluadas fueron altura del explante (cm), número de brotes y número de hojas, a los 60 y 120 días de cultivo. Tanto el uso de Zeatina como kinetina en el medio de cultivo WP permiten la multiplicación in vitro de M. rupestris. Sin embargo, la adición de Zeatina favorece el desarrollo de las plantas con características fenotípicas adecuadas para la micropropagación. Atendiendo a los resultados se propone utilizar el medio de cultivo A (WP+kinetina 0.5 mg l-1)
Harry Van Onckelen - One of the best experts on this subject based on the ideXlab platform.
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in situ localisation of cytokinins in the shoot apical meristem of sinapis alba at floral transition
Planta, 2002Co-Authors: Annie Jacqmard, W. Dewitte, Harry Van Onckelen, Nathalie Detry, Georges BernierAbstract:In plants of Sinapis alba L. induced to flower by one long day (LD), previous work showed that the phloem sap feeding the shoot apex is enriched in cytokinins of the isopentenyladenine (iP)-type between 9 and 25 h after start of the LD [P. Lejeune et al. (1994) Physiol Plant 90:522–528]. We have checked the hypothesis that the cytokinin content of the shoot apical meristem (SAM) should increase in response to floral induction by one LD using histoimmunolocalisation techniques and rabbit antiserum against isopentenyladenosine or Zeatin riboside. The free bases iP and Zeatin are present only in apical tissues containing dividing cells. At 30 h after the start of an inductive LD, a markedly increased iP immune reaction is observed in SAM tissues while the level of Zeatin is not modified. Our results are in line with the data obtained by analysis of phloem sap.
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Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes Due to Light/Dark Treatment
Plant physiology, 1999Co-Authors: Eva Benková, Václav Motyka, Erwin Witters, Walter Van Dongen, Jan Kolar, Bretislav Brzobohatý, Harry Van Onckelen, Ivana MacháčkováAbstract:Although cytokinins (CKs) affect a number of processes connected with chloroplasts, it has never been rigorously proven that chloroplasts contain CKs. We isolated intact chloroplasts from tobacco ( Nicotiana tabacum L. cv SR1) and wheat ( Triticum aestivum L. cv Ritmo) leaves and determined their CKs by liquid chromatography/tandem mass spectroscopy. Chloroplasts from both species contained a whole spectrum of CKs, including free bases (Zeatin and isopentenyladenine), ribosides (Zeatin riboside, and isopentenyladenosine), ribotides (isopentenyladenosine-5′-monophosphate, Zeatin riboside-5′-monophosphate, and dihydroZeatin riboside-5′-monophosphate), and N -glucosides (Zeatin- N 9 -glucoside, dihydroZeatin- N 9 -glucoside, Zeatin- N 7 -glucoside, and isopentenyladenine- N -glucosides). In chloroplasts there was a moderately higher relative amount of bases, ribosides, and ribotides than in leaves, and a significantly increased level of N 9 -glucosides of Zeatin and dihydroZeatin. Tobacco and wheat chloroplasts were prepared from leaves at the end of either a dark or light period. After a dark period, chloroplasts accumulated more CKs than after a light period. The differences were moderate for free bases and ribosides, but highly significant for glucosides. Tobacco chloroplasts from dark-treated leaves contained Zeatin riboside- O -glucoside and dihydroZeatin riboside- O -glucoside, as well as a relatively high CK oxidase activity. These data show that chloroplasts contain a whole spectrum of CKs and the enzymatic activity necessary for their metabolism.
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Dynamics of Cytokinins in Apical Shoot Meristems of a Day-Neutral Tobacco during Floral Transition and Flower Formation
Plant physiology, 1999Co-Authors: W. Dewitte, Miroslav Strnad, Erwin Witters, Adriana Chiappetta, Abdelkrim Azmi, Jacques Rembur, Michelle Noin, Dominique Chriqui, Harry Van OnckelenAbstract: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.
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Levels of endogenous cytokinins, indole-3-acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY-2 cells
FEBS letters, 1996Co-Authors: Pascale Redig, Orit Shaul, Dirk Inzé, Marc Van Montagu, Harry Van OnckelenAbstract:Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY-2 cell suspension cultures. Sixteen different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) were extracted using solid-phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (Zeatin- and dihydroZeatin-type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including Zeatins N- and O-glucosides, remained low, suggesting that the increased amounts of their corresponding non-glucosylated from resulted from de novo synthesis. These findings suggest that Zeatin- and dihydroZeatin-type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.
David W. S. Mok - One of the best experts on this subject based on the ideXlab platform.
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DEVELOPMENT OF TRANSGENIC TOBACCO HARBORING A Zeatin O-GLUCOSYLTRANSFERASE GENE FROM PHASEOLUS
2014Co-Authors: Ruth C. Martin, David W. S. Mok, Harry A. Van Onckelen, Machteld C. MokAbstract:Zeatin and its derivatives are major constituents of higher plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a Zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) and an inducible (Tet) promoter. The presence of the transgene resulted in elevated enzyme production and conversion of exogenous Zeatin to its O-glucoside, confirming the expression of the ZOG1 gene in transgenic plants. Endogenous O-glucosylZeatin was increased from less than 1 pmol per g fresh weight in leaves and roots of controls to 26 and 68 pmol per g fresh weight in leaves and roots of 35S-ZOG1 transformants, respectively. In cytokinin/auxin interaction experiments, Tet-ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher Zeatin concentrations for the formation of shoots and callus than the controls. In 35S-ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus, increased Zeatin O-glucosylation in detached, cytokinin-dependent tissues leads to a shift in the response to exogenous Zeatin indicative of cytokinin sequestering. In whole plants the effect can simulate a reduction or a rise in cytokinin activity depending on the tissue and stage of development. The use of tissue- and stage-specific promoters in the future will allow more precise analyses and targeted growth alterations. Key words: cytokinin; transformation; Zeatin metabolism; root formation; apical dominance
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topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin o glucosyltransferase with position specificity related to receptor recognition
Plant Physiology, 2005Co-Authors: Machteld C. Mok, Petre I Dobrev, Ruth C. Martin, Hitoshi Sakakibara, Radomira Vankova, Keiko Yonekurasakakibara, David W. S. MokAbstract:Glucosides of trans-Zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-Zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-Zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-Zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-Zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.
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o glucosylation of cis Zeatin in maize characterization of genes enzymes and endogenous cytokinins
Plant Physiology, 2003Co-Authors: Yeonjin K Veach, David W. S. Mok, Ruth C. Martin, Jiri Malbeck, Radomira Vankova, Machteld C. MokAbstract:trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-Zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize ( Zea mays ) gene ( cisZOG1 ) encoding an O -glucosyltransferase specific to cis-Zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922–5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2 , and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K m values of 46 and 96 μm, respectively, for cis-Zeatin and a pH optimum of 7.5. Other cytokinins, including N 6 -(Δ 2 -isopentenyl)adenine, trans-Zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-Zeatin riboside, and their O -glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O -glucoside of cis-Zeatin riboside. The results are a clear indication that O -glucosylation of cis-Zeatin is a natural metabolic process in maize. Whether cis-Zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.
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a maize cytokinin gene encoding an o glucosyltransferase specific to cis Zeatin
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Ruth C. Martin, Machteld C. Mok, Jeffrey E Habben, David W. S. MokAbstract:Zeatin is a naturally occurring cytokinin. Biosynthesis and metabolism studies of Zeatin have been directed mostly at the trans isomer, although cis-Zeatin and its riboside occur as major components in some plant species. It is not known whether parallel regulatory pathways exist for the two isomers. Based on the sequence of the gene ZOG1 encoding a trans-Zeatin O-glucosyltransferase from Phaseolus (EC 2.4.1.203), a cis-Zeatin-specific O-glucosyltransferase was isolated from maize. This gene, cisZOG1, contains an ORF of 1,401 nucleotides encoding a protein of 51.1 kDa with 41% identity to the Phaseolus ZOG1 protein. Unexpectedly, the maize enzyme recognizes as substrates cis-Zeatin and UDP-glucose but not cis-ribosylZeatin, trans-Zeatin, or trans-ribosylZeatin. This finding indicates the existence of cis-specific regulatory elements in plants and suggests that cis-Zeatin and derivatives may be more important in cytokinin homeostasis than currently recognized.
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isolation of a cytokinin gene zog1 encoding Zeatin o glucosyltransferase from phaseolus lunatus
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Ruth C. Martin, Machteld C. Mok, David W. S. MokAbstract:Zeatin is the most active and ubiquitous of the naturally occurring cytokinins. The O-glucoside of Zeatin, found in all plants examined, is considered to be important in cytokinin transport, storage, and protection against cytokinin oxidases. The enzyme UDPglucose:Zeatin O-glucosyltransferase (EC 2.4.1.203) was previously isolated from Phaseolus lunatus seeds. Immunoscreening of an expression library with monospecific antibody resulted in the isolation of a cDNA encoding the enzyme. The recombinant protein efficiently converts labeled Zeatin to O-glucosylZeatin and has properties similar to the native enzyme. The cDNA of 1.5 kb contains an ORF encoding a 51.4-kDa polypeptide of 459 amino acids. The sequence is unique based on a blast search of data bases. The genomic sequence, isolated with PCR using specific primers based on the cDNA sequence, does not contain introns. The cloning of this gene provides the tools for further study of the regulation of cytokinin metabolism and analysis of the precise role of O-glucosylZeatin in plant development.
Machteld C. Mok - One of the best experts on this subject based on the ideXlab platform.
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DEVELOPMENT OF TRANSGENIC TOBACCO HARBORING A Zeatin O-GLUCOSYLTRANSFERASE GENE FROM PHASEOLUS
2014Co-Authors: Ruth C. Martin, David W. S. Mok, Harry A. Van Onckelen, Machteld C. MokAbstract:Zeatin and its derivatives are major constituents of higher plant cytokinins. Metabolic steps modifying the isoprenoid side chain, such as O-glycosylation, are expected to have a direct bearing on cytokinin-mediated processes. To examine this possibility, transgenic tobacco plants were generated harboring a gene (ZOG1) encoding a Zeatin O-glucosyltransferase from Phaseolus lunatus under the control of a constitutive (35S) and an inducible (Tet) promoter. The presence of the transgene resulted in elevated enzyme production and conversion of exogenous Zeatin to its O-glucoside, confirming the expression of the ZOG1 gene in transgenic plants. Endogenous O-glucosylZeatin was increased from less than 1 pmol per g fresh weight in leaves and roots of controls to 26 and 68 pmol per g fresh weight in leaves and roots of 35S-ZOG1 transformants, respectively. In cytokinin/auxin interaction experiments, Tet-ZOG1 leaf discs, in the presence of tetracycline, required 10-fold higher Zeatin concentrations for the formation of shoots and callus than the controls. In 35S-ZOG1 plants, developmental changes included adventitious root formation on the lower stems, shorter stature, and axillary shoot growth. Thus, increased Zeatin O-glucosylation in detached, cytokinin-dependent tissues leads to a shift in the response to exogenous Zeatin indicative of cytokinin sequestering. In whole plants the effect can simulate a reduction or a rise in cytokinin activity depending on the tissue and stage of development. The use of tissue- and stage-specific promoters in the future will allow more precise analyses and targeted growth alterations. Key words: cytokinin; transformation; Zeatin metabolism; root formation; apical dominance
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topolins and hydroxylated thidiazuron derivatives are substrates of cytokinin o glucosyltransferase with position specificity related to receptor recognition
Plant Physiology, 2005Co-Authors: Machteld C. Mok, Petre I Dobrev, Ruth C. Martin, Hitoshi Sakakibara, Radomira Vankova, Keiko Yonekurasakakibara, David W. S. MokAbstract:Glucosides of trans-Zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-Zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-Zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-Zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-Zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results.
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o glucosylation of cis Zeatin in maize characterization of genes enzymes and endogenous cytokinins
Plant Physiology, 2003Co-Authors: Yeonjin K Veach, David W. S. Mok, Ruth C. Martin, Jiri Malbeck, Radomira Vankova, Machteld C. MokAbstract:trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-Zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize ( Zea mays ) gene ( cisZOG1 ) encoding an O -glucosyltransferase specific to cis-Zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922–5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2 , and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K m values of 46 and 96 μm, respectively, for cis-Zeatin and a pH optimum of 7.5. Other cytokinins, including N 6 -(Δ 2 -isopentenyl)adenine, trans-Zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-Zeatin riboside, and their O -glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O -glucoside of cis-Zeatin riboside. The results are a clear indication that O -glucosylation of cis-Zeatin is a natural metabolic process in maize. Whether cis-Zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.
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a maize cytokinin gene encoding an o glucosyltransferase specific to cis Zeatin
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Ruth C. Martin, Machteld C. Mok, Jeffrey E Habben, David W. S. MokAbstract:Zeatin is a naturally occurring cytokinin. Biosynthesis and metabolism studies of Zeatin have been directed mostly at the trans isomer, although cis-Zeatin and its riboside occur as major components in some plant species. It is not known whether parallel regulatory pathways exist for the two isomers. Based on the sequence of the gene ZOG1 encoding a trans-Zeatin O-glucosyltransferase from Phaseolus (EC 2.4.1.203), a cis-Zeatin-specific O-glucosyltransferase was isolated from maize. This gene, cisZOG1, contains an ORF of 1,401 nucleotides encoding a protein of 51.1 kDa with 41% identity to the Phaseolus ZOG1 protein. Unexpectedly, the maize enzyme recognizes as substrates cis-Zeatin and UDP-glucose but not cis-ribosylZeatin, trans-Zeatin, or trans-ribosylZeatin. This finding indicates the existence of cis-specific regulatory elements in plants and suggests that cis-Zeatin and derivatives may be more important in cytokinin homeostasis than currently recognized.
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isolation of a cytokinin gene zog1 encoding Zeatin o glucosyltransferase from phaseolus lunatus
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Ruth C. Martin, Machteld C. Mok, David W. S. MokAbstract:Zeatin is the most active and ubiquitous of the naturally occurring cytokinins. The O-glucoside of Zeatin, found in all plants examined, is considered to be important in cytokinin transport, storage, and protection against cytokinin oxidases. The enzyme UDPglucose:Zeatin O-glucosyltransferase (EC 2.4.1.203) was previously isolated from Phaseolus lunatus seeds. Immunoscreening of an expression library with monospecific antibody resulted in the isolation of a cDNA encoding the enzyme. The recombinant protein efficiently converts labeled Zeatin to O-glucosylZeatin and has properties similar to the native enzyme. The cDNA of 1.5 kb contains an ORF encoding a 51.4-kDa polypeptide of 459 amino acids. The sequence is unique based on a blast search of data bases. The genomic sequence, isolated with PCR using specific primers based on the cDNA sequence, does not contain introns. The cloning of this gene provides the tools for further study of the regulation of cytokinin metabolism and analysis of the precise role of O-glucosylZeatin in plant development.
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auxin indole 3 acetic acid gibberellic acid ga3 abscisic acid aba and cytokinin Zeatin production by some species of mosses and lichens
Turkish Journal of Botany, 2002Co-Authors: Nuray ErgunAbstract:In this study, the levels of endogenous free, bound and total auxin (Indole-3-acetic acid, IAA), gibberellic acid (GA3), abscisic acid (ABA) and cytokinin (Zeatin) were examined in some species of mosses and lichens. For determination of the levels of these plant growth regulators, spectrophotometry was used. Our findings show that the mosses and lichens used in this study produce the plant growth regulators IAA, GA3, ABA and Zeatin.
