The plant hormone cytokinin plays essential roles in many aspects of growth and development. The cytokinin signal is transmitted by a multistep phosphorelay to the members of two functionally antagonistic classes of Arabidopsis response regulators (ARRs): the type-B ARRs (response activators) and type-A ARRs (negative-feedback regulators). Previous studies have shown that mutations in AXR1, encoding a subunit of the E1 enzyme in the related to ubiquitin (RUB) modification pathway, leads to decreased cytokinin sensitivity. This research shows that the cytokinin resistance of axr1 seedlings is suppressed by loss-of-function of type-A ARRs and that the cytokinin resistance caused by ectopic expression of ARR5, a type-A ARR family member, is enhanced in axr1 background. Based on the established role of the RUB pathway in ubiquitin-dependent proteolysis, these data suggested that AXR1 promotes the cytokinin response by facilitating type-A ARR degradation. Indeed, both genetic (axr1 mutants) and chemical (MLN4924) suppression of RUB E1 increased ARR5 stability, suggesting that the ubiquitin ligase that promotes ARR5 proteolysis requires RUB modification for optimal activity. In addition, ARR1, a type-B ARR family member, also accumulated in the axr1 mutant background, suggesting that AXR1 regulates primary cytokinin signaling at multiple levels.

Cytokinins (CKs) are one of the most important group of phytohormones influencing many processes throughout the whole plant. As many processes are regulated both by the light and phytohormones, the first part of this work has been focused on evaluation of diurnal rhytmicity in levels of cytokinins and other cooperating hormones like auxin (indol-3-acetic acid, IAA), abscisic acid (ABA) and polyamines (PA). The changes in activity of selected enzymes participating in metabolism of the above mentioned phytohormones were followed as well. Diurnal variation of phytohormones was tested in tobacco leaves (Nicotiana tabacum L. cv. Wisconsin 38) grown under a 16/8 h (light/dark) period. The main peak of the physiologically active forms of CKs, found after the middle of the light period, coincided well with the maximum of IAA and PA levels and with activity of the corresponding enzymes. The achieved data indicate that metabolism of CKs, IAA and PAs is tightly regulated by the circadian clock. The other part of the study has been focused on changes in the contents of CKs, IAA and ABA in transgenic tobacco plants with altered cytokinin metabolism, achieved via the over-expression of particular enzymes participating in CK metabolism (biosynthesis, degradation and reversible conjugation). As CKs are known to be...

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This diploma thesis was elaborated on the theme "The study of factors influencing service tree (Sorbus domestica L.) micropropagation". The experiments were carried out in in vitro laboratory of the Mendeleum - Institute of Genetics and Plant Breeding of the Faculty of Horticulture in Lednice. The aim of this work was to acquire and improve the knowledge of in vitro cultivation of serviceberry and the genus Sorbus L. in general. The trees growing in experimental land of the Faculty of Horticulture MZLU in Lednice were selected as experimental material. The composition of cultivation medium was verified for plant growth and multiplication purposes. The influence of two different cytokinins BA (benzyl-adenine) and zeatin was evaluated. The shoot number and shoot length were measured. The highest multiplication rate evaluated by the origin of new formed shoots was accomplished on media supplemented with BA (0.1 mg.l-1). Nevertheless the shortest height of shoots was measured on this media. It was valid for both cytokinins that number of shoots was increasing with increasing cytokinin concentration whilst the shoot height was decreasing.

Made available in DSpace on 2014-06-11T19:26:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-04Bitstream added on 2014-06-13T20:55:05Z : No. of bitstreams: 1 silva_smg_me_botfca.pdf: 943123 bytes, checksum: e1f346a000e6f16cebed0347a5eb69fe (MD5) / Plântulas provenientes da germinação de sementes in vitro da bromélia Aechmea blanchetiana foram cultivadas em meio líquido para o estudo do crescimento sob a influência de diferentes reguladores vegetais. Os tratamentos consistiram da combinação de concentrações (mg L-1) de BAP (0,2), Kt (0,2), NAA (0,1) e IBA (0,1) adicionados aos meios de cultivo líquido MS. As plântulas foram cultivadas sob fotoperíodo de 16 horas e temperatura de 25 ºC ± 2, durante 90 dias e subcultivos a cada 15 dias. O delineamento experimental adotado foi o inteiramente casualizado para todas as variáveis consideradas com 9 tratamentos e 4 repetições, totalizando 36 parcelas, sendo cada parcela composta por 4 plântulas perfazendo um total de 144 plântulas. Foram medidas e analisadas as seguintes características: comprimento da maior folha, número de folhas, número de raízes, comprimento da maior raiz, massa fresca e seca de plântulas e raízes. O BAP promoveu o melhor resultado para as variáveis número de raízes e comprimento da maior raiz. Os tratamentos com NAA e IBA apresentaram o melhor resultado quanto ao comprimento da maior folha, gerando ambos melhores desempenho para a massa de matéria fresca de plântulas / Seedlings from in vitro seed germination of the bromeliad Aechmea blanchetiana were cultured in liquid medium to study growth under influence of different plant growth regulators. Treatments were combined concentrations (mg L-1) of BAP (0.2), Kinetin (0.2), NAA (0.1) and IBA (0.1) added to MS liquid culture media. Seedlings were cultured under 16h photoperiod and 25 ºC ± 2 for 90 days and subcultured every 15 days. Experimental design was completely randomized for all variables, with 9 treatments and 4 replicates totaling 36 plots, each one composed of 4 seedlings, 144 in total. Length of the largest leaf, number of leaves, number of roots, length of the largest root, and fresh and dry matter of seedlings and roots were measured and analyzed. BAP led to the best results for number of roots and length of the largest root. Treatments with NAA and IBA led to the best values as to length of the largest leaf, both resulting in higher values of seedling fresh matter

Worldwide production of potatoes is in excess of 350 million metric tonnes per annum, of which 60% is intended for human consumption. Since the period of tuber dormancy before tuber buds sprout is usually shorter than the optimal market storage period, control of sprouting is essential. To prolong dormancy, tubers are either stored at low temperatures and/or are treated with chemical sprout inhibitors, the use of which subject to increasing scrutiny in the European Union due to their impact upon the environment. Cytokinins, a group of plant growth regulators, are known to play a central role in tuber bud sprouting and tuber initiation from stolon tips in Solanum tuberosum L. although it is unclear when and how cytokinins act to regulate dormancy. The interconversion of cytokinins is incompletely understood. Enzymes identified to date have higher affinities for aminopurines than their cytokinin equivalents. A novel cytokinin binding protein Solanum tuberosum Cytokinin Phosphorylase 1 (StCKP1), has been identified in tuberising stolon tips which shares regions of homology with members of the nucleosidase and phosphoribosyl transferase family. The composition of cytokinin N9 conjugates in tuber bud and surrounding tissue is known to change on transition from a dormant state, with an increase in base and riboside types observed. Analysis of transcripts indicates an increase in abundance of StCKP1 on tuberisation of stolon tips, and high abundance in the periderm of dormant tubers. Analysis of protein abundance by immunoblotting echoes this finding and indicates StCKP1 begins to accumulate in stolon tips shortly before tuberisation, matching binding activity. Transgenic analysis of the cytological reporter gene uidA under the control of two identified promoter regions indicates StCKP1 is expressed predominantly in tuber tissue. Analysis of StCKP1 activity by HPLC and LC-MS-MS shows that StCKP1 catalyses the interconversion of free base and riboside. KMs determined for cytokinin and aminopurine substrates indicate that StCKP1 has a higher affinity for cytokinin substrates and, of these cytokinins, displays a higher affinity for the free base catalysing ribosylation of the N9 to form the corresponding riboside. Desiree cultivars over-expressing StCKP1 under the CaMV 35S promoter exhibited an increased rate of tuberisation of stolon tips and an increase in the length of the dormant period following lifting. Over-expression of StCKP1 was found in particular to increase the chill sensitive period of dormancy, confirming results of StCKP1 knock-down by RNAi. Transcript abundance of StCKP1 at tuberisation in other cultivars including King Edward and Maris Peer was found to correlate with the dormancy characteristics prescribed by the European Cultivated Potato Database and the British potato variety database.

Orientador: Gisela Ferreira / Banca: Andrea Maria Teixeira Fontes / Banca: João Domingo Rodrigues / Resumo: A espécie Passiflora setacea DC conhecida como maracujá-do-sono é nativa do cerrado podendo ocorrer na caatinga e em áreas de transição como o semi-árido e nortemineiro e tem importante potencial como porta-enxertos para espécies comerciais de maracujá. Desse modo o presente trabalho teve como objetivo avaliar condições de luz, temperaturas e reguladores vegetais na germinação de sementes de P. Setacea. O trabalho foi desenvolvido em duas etapas. Na primeira avaliou-se várias concentrações de diferentes reguladores em condição de luz e escuro. O delineamento experimental foi inteiramente casualizado com 66 tratamentos, 5 repetições de 25 sementes por parcela, em esquema fatorial 3x11x2 (reguladores X concentrações X luz e escuro). Os reguladores empregados foram: o acído giberelico GA3; GA4+7 + N-(fenilmetil)- aminopurina e, GA3 somado a GA4+7 + N-(fenilmetil)- aminopurina, nas seguintes concentrações: 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg L-1 , na presença e ausência de luz. Na segunda etapa, avaliou-se diferentes temperaturas, com diferentes reguladores, em condições de luz e escuro. O delineamento experimental foi inteiramente casualizado com 56 tratamentos, 5 repetições de 25 sementes por parcela em esquema fatorial 7x4x2 (temperaturas X reguladores X luz e escuro). As temperaturas empregadas foram: 20°C, 25°C, 30°C, 35°C, 40°C, 30/20°C e 20/30°C (16/8 horas respectivamente). Os reguladores utilizados foram: 100 mgL-1 de GA3, 100 mgL-1 de GA4+7 +N-(fenilmetil)- aminopurina, a mistura de 100 mgL-1 de GA3 + 100 mgL-1 de GA4+7 + N-(fenilmetil)- aminopurina i.a. e água destilada. Foram calculadas as seguintes variáveis: porcentagem de germinação, índice de sincronização e índice de velocidade de germinação. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Tukey a 5% de probabilidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The species Passiflora setacea DC known as fruit-of-sleep is native to the cerrado may occur in the caatinga and in areas of transition such as semi-arid and norte-mineiro and has significant potential as rootstock for commercial species of passion fruit. Thus the present study was to evaluate conditions of light, temperature and plant growth regulators in seed germination of P. Setacea. The study was conducted in two stages. The first focuses on various concentrations of different regulators on condition of light and dark. The experimental design was completely randomized with 66 treatments, 5 replicates of 25 seeds per plot in a factorial 3x11x2 (regulators X concentrations X light and dark). The regulators employed were: the gibberellic acid GA3; GA4+7+N-(phenylmethyl) - aminopurina and GA3 plus GA4+7+ N-(phenylmethyl) - aminopurina in the following concentrations: 0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg L-1 in the presence and absence of light. In the second step, it was evaluated different temperatures, with different regulators, under conditions of light and dark. The experimental design was completely randomized to 56 treatments, 5 replicates of 25 seeds per plot in a factorial 7x4x2 (temperature X regulators X light and dark). The temperatures used were: 20°C, 25°C, 30°C, 35°C, 40°C, 30/20°C and 20/30°C (16 / 8 hours respectively). The regulators used were: 100 mgL-1 GA3, 100 mgL-1 of GA4+7+ N-(phenylmethyl) - aminopurina, the mixture of 100 mgL-1 GA3 + 100 mgL-1 of GA4+7+ N - (phenylmethyl) - aminopurina and distilled water. We calculated the following variables: percentage of germination, rate of synchronization and speed of germination index. Data were submitted to analysis of variance and averages compared by Tukey test at 5% probability. It is concluded that the interaction of GA3 and / or GA4+7+ N-(phenylmethyl) - aminopurina is effective... (Complete abstract click electronic access below) / Mestre

Orientador: Claudia Regina Baptista Haddad / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T21:10:07Z (GMT). No. of bitstreams: 1 Chone_RosanaMarySartor_M.pdf: 46520912 bytes, checksum: 140ac9102b951f96df903dee18f16c8a (MD5) Previous issue date: 2015 / Resumo: O cultivo do cafeeiro é de grande importância econômica mundial. O consumo crescente de café arábica tem levado os pesquisadores a buscar variedades que produzam bebidas de melhor qualidade e com baixos teores de cafeína. Os programas de melhoramento genético concentram esforços na busca destas características com destaque para cultivares da espécie Coffea arabica, a mais cultivada no mundo. A embriogênese somática é importante para manter características selecionadas em programas de melhoramento genético e produzir os indivíduos selecionados em grande escala, além de contribuir para estudos fisiológicos e moleculares. A embriogênese somática direta apresenta vantagens pela redução de insumos e mão de obra, devido à eliminação da fase calogênica, porém, apresenta baixa eficiência, quando comparada com a via indireta, para a espécie C. arabica. Apesar da importância da embriogênese somática direta, nada se sabe sobre os fatores que controlam a sua ocorrência em genótipos de C. arabica e tampouco há estudos com utilização de brassinosteróides na fase de indução desse tipo de embriogênese in vitro. O brassinosteróide, 24-epibrassinolídeo, foi utilizado isoladamente e em conjunto com uma citocinina, N6-(2-isopentenyl) adenina (2-iP), para avaliar seus efeitos sobre a embriogênese somática direta e verificar possíveis alterações endógenas no metabolismo desses hormônios durante a indução da embriogênese em explantes foliares da cultivar Mundo Novo de Coffea arabica. Os explantes foram cultivados em meio de cultura MS modificado, adicionado de 2-iP a 10 µM, acrescido ou não de 24-epibrassinolídeo. Foram utilizadas as seguintes concentrações do brassinosteróide: 0,01 µM, 0,10 µM e 1 µM, em dois experimentos realizados em diferentes épocas do ano. Análises de microscopia de luz e microscopia eletrônica de varredura foram realizadas para compreensão dos eventos anatômicos e morfológicos da via de embriogênese relacionada às diferentes combinações hormonais praticadas. Avaliações de variáveis qualitativas e quantitativas dos explantes foliares dos dois experimentos foram realizadas in vitro durante 130 e 240 dias. Na tentativa de compreender melhor a participação dos brassinosteróides na via de embriogênese somática, explantes foliares induzidos apenas com a citocinina, 2-iP, foram submetidos a análises de Ressonância Magnética Nuclear (RMN). Os embriões somáticos obtidos tanto no tratamento com uso exclusivo de citocinina, como no tratamento com citocinina associada ao 24-epibrassinolídeo foram cultivados em meio de cultura para germinação e desenvolveram-se em plântulas. A aplicação do brassinosteróide levou à iniciação do processo de embriogênese somática direta, promovendo a formação de estruturas embriogênicas. Sua aplicação conjunta com citocinina aumentou o número de embriões, o número de explantes com embriões, levou à formação de embriões em condição na qual apenas a aplicação de citocinina foi insuficiente e acelerou a via embriogênica. Promoveu o desenvolvimento de embriões mais bem definidos, com características meristemáticas típicas. O aumento de picos na região do espectro de RMN, característico de moléculas esteroidais, coincidente com o período em que ocorre a embriogênese somática direta, pode ser indicativo da participação de brassinosteróides endógenos durante esse processo. Este trabalho abre perspectivas para a utilização de brassinosteróides em embriogênese somática direta em processos produtivos de C. arabica / Abstract: Coffee cultivation is of worldwide economic importance. The increasing consumption of coffee has led to search for varieties that produce better quality beverages with lower caffeine contents. Genetic improvement have concentrated their efforts on the search for these characteristics with emphasis on cultivars of the species Coffea arabica, the most cultivated in the world. Somatic embryogenesis is important to maintain selected characteristics in genetic improvement and produce selected individuals on a large scale, as well as contributing to physiological and molecular studies. Direct somatic embryogenesis shows the advantages of reduced consumables and less manual labor, due to elimination of the callus phase, but is inefficient with the species C. arabica when compared with the indirect pathway. Despite the importance of direct somatic embryogenesis, nothing is known about factors controlling its occurrence in C. arabica genotypes, nor are there any studies on use of brassinosteroids in the in vitro induction of this type of embryogenesis. The brassinosteroid 24-epibrassinolide was used alone and together with cytokinin N6-(2-isopentenyl) adenine (2-iP) to evaluate its effects on direct somatic embryogenesis and verify endogenous alterations in metabolism of these hormones during induction of embryogenesis in foliar explants of cultivar Mundo Novo of species Coffea Arabica. The explants were cultivated in modified MS culture medium with the addition of 10 µM 2-iP, with or without 24-epibrassinolide. The following concentrations of brassinosteroid were used: 0.01 µM, 0.10 µM and 1 µM, in two experiments carried out at different times of the year. Light microscopy and scanning electronic microscopy analyses were carried out in order to understand anatomic and morphologic events of embryogenic pathway as related to different hormone combinations applied. The qualitative and quantitative variables of the foliar explants were evaluated in the two experiments at points in time from 130 and 240 days. In an attempt to better understand the participation of the brassinosteroids in the somatic embryogenesis pathway, foliar explants induced only by the cytokinin 2-iP were submitted to nuclear magnetic resonance (RMN) analyses. The somatic embryos obtained both using the treatment with the exclusive use of cytokinin and in treatment with cytokinin associated with 24-epibrassinolide were cultivated in a culture medium for germination and developed into plant embryos. The application of brassinosteroid led to initiation of direct somatic embryogenesis process, promoting the formation of embryogenic structures. Its application together with cytokinin increased number of embryos, the number of explants with embryos, led to formation of embryos in a condition in which only application of cytokinin was insufficient and accelerated embryogenic pathway. It also promoted development of much better defined embryos with typically meristematic characteristics. The increase in peaks in the region of the RMN spectrum, characteristic of steroidal molecules, coinciding with the period in which direct somatic embryogenesis occurred, could be an indication of participation of endogenous brassinosteroids during the process. This work widens the perspectives for the use of brassinosteroids in direct somatic embryogenesis for productive processes with C. arabica / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

Plants had to evolve, due to their sessile growth habit, a complex system of defence against adverse environmental conditions. Plant abiotic stress responses are regulated by plant hormones, especially by cytokinins (CKs) and abscisic acid (ABA). In order to evaluate the effect of enhanced stress tolerance on the dynamics of hormones (CKs, auxin and ABA) as well as polyamine levels during the drought and/or heat stress (HS) progression, response of tobacco plants with enhanced level of osmolyte proline was compared with the corresponding wild-type. HS (40řC) caused a transient increase in bioactive CK content, accompanied by decrease of ABA, which indicated stimulation of transpiration, important mechanism to cool down the leaf temperature at the early phase of HS. Simultaneously, a transient increase in spermidine and spermine levels was found, more profound in proline-over-producing transformant. The activities of particular polyamine metabolic enzymes correlated well with their content. Drought stress resulted in the establishment of a gradient of bioactive CKs in favour of the upper leaves, crucial for their preferential protection. Application of HS at the end of drought period strongly enhanced the stress severity, mainly due to additional water loss caused by enhanced transpiration....

Charles University in Prague, Faculty of Science Department of Experimetnal Plant Biology Ph.D. study program: Plant physiology and anatomy Abstract Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level. Zuzana Lubovská Supervisor: RNDr. Naďa Wilhelmová, CSc. Supervisor-consultant: RNDr. Helena Štorchová, CSc. Praha, 2015 The response of the antioxidant enzyme system to drought, heat and a combination of these stresses was followed in tobacco plants overexpressing cytokinin oxidase/dehydrogenase1 (CKX1) under the root-specific WRKY6 promoter (W6:CKX1 plants) and under the constitutive 35S promoter (35S:CKX1 plants) and in the corresponding wild type (WT). CKX1 over-producing lines developed a bigger root system, which contributed to their stress tolerance. The high stress tolerance of 35S:CKX1 plants was also associated with a dwarf shoot phenotype and changed leaf morphology. The genes for chloroplastic antioxidant enzymes, stroma ascorbate peroxidase (sAPX), thylakoid ascorbate peroxidase (tAPX) and chloroplastic superoxide dismutase (FeSOD), which are responsible for scavenging of reactive oxygen species produced via electron transfer during photosynthesis, were all strongly transcribed in control conditions. All the tested stresses down-regulated expression of these genes...