A cDNA sequence representing a <i> Brassica carinata</i> gene the expression of which is induced by copper chloride treatment, was isolated from a library constructed with mRNA from treated leaves, and designated CIL1 (COPPER CHLORIDE INDUCED in LEAVES). A Basic Local Alignment Search Tool search revealed that CIL1 has similarities to an auxin-induced gene, AIR12 from <i>Arabidopsis thaliana</i>. Southern blot analysis of CIL1 in <i>B. carinata, B. nigra</i> and <i>B. oleracea</i> indicated that it is a member of a small multigene family. Antisense CIL1 transgenic plants were generated to investigate the function of CIL1, and the resulting transformants displayed increased secondary branching suggesting that CIL1 has a role in regulating hormone content or plant architecture. Results of induction studies indicate that the auxin analog a-naphthalene acetic acid, the cytokinin 6-benzylaminopurine, and +/- abscisic acid increase expression of CIL1. Seven CIL1 antisense lines were grown to the T4 generation and were confirmed homozygous. Analysis of CIL1 expression using real-time quantitative RT-PCR showed reduced expression in every examined line. Transgenic plants produced many leaves at the lateral meristems indicating a release of apical dominance. Additionally, the concentrations of auxins, cytokinins, and abscisic acid were altered in the roots and stems of transgenic plants compared to non-transformed plants. Therefore, CIL1 has a role in regulating hormone content that affects lateral meristem activity, apical dominance, and leaf production.

A cDNA sequence representing a <i> Brassica carinata</i> gene the expression of which is induced by copper chloride treatment, was isolated from a library constructed with mRNA from treated leaves, and designated CIL1 (COPPER CHLORIDE INDUCED in LEAVES). A Basic Local Alignment Search Tool search revealed that CIL1 has similarities to an auxin-induced gene, AIR12 from <i>Arabidopsis thaliana</i>. Southern blot analysis of CIL1 in <i>B. carinata, B. nigra</i> and <i>B. oleracea</i> indicated that it is a member of a small multigene family. Antisense CIL1 transgenic plants were generated to investigate the function of CIL1, and the resulting transformants displayed increased secondary branching suggesting that CIL1 has a role in regulating hormone content or plant architecture. Results of induction studies indicate that the auxin analog a-naphthalene acetic acid, the cytokinin 6-benzylaminopurine, and +/- abscisic acid increase expression of CIL1. Seven CIL1 antisense lines were grown to the T4 generation and were confirmed homozygous. Analysis of CIL1 expression using real-time quantitative RT-PCR showed reduced expression in every examined line. Transgenic plants produced many leaves at the lateral meristems indicating a release of apical dominance. Additionally, the concentrations of auxins, cytokinins, and abscisic acid were altered in the roots and stems of transgenic plants compared to non-transformed plants. Therefore, CIL1 has a role in regulating hormone content that affects lateral meristem activity, apical dominance, and leaf production.

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Phytohormones act as signals to regulate plant growth and development by modulation of gene expression in response to internal developmental cues or external environmental stimuli, such as light and pathogen infection. There are five major classes of phytohormones, viz. auxins, cytokinins, gibberellins, ethylene and abscisic acid. Of these, cytokinins, 6N substituted adenine derivatives, are of special importance owing to their possible diverse roles in plant growth and development. They induce cell division, cell expansion in cotyledon, chloroplast and etioplast development, suppression of apical dominance and senescence, and differentiation of in vitro cultured cells. However, very little is known about the mechanism of cytokinin action at the molecular level. Cytokinins have been demonstrated to modulate the expression of genes coding for several enzymes including nitrate reductase, ribulose-l,5-bisphosphate carboxylase, RNA polymerase I, and pathogenesis-related (PR) enzymes, i.e. chitinases and β-1,3- glucanases. One of the important questions regarding cytokinin regulation of enzyme activities and/or the accumulation of their corresponding proteins and mRNAs is how the cytokinin signal is transduced. There is considerable evidence from earlier reports demonstrating that pathogens alter hormone physiology of the host plant and it has been proposed that the infection-associated enzyme changes might be mediated by phytohormones. In the present study, two PR enzymes, viz. cucumber chitinase and β-l,3-glucanase, have been chosen to examine the mode of regulation of their gene expression by cytokinins, including the identification of cytokinin signal transduction components. Plant chitinases and glucanases are important enzymes in plant defense mechanisms against fungal pathogens as they degrade the major fungal cell wall components, chitin and β-1,3-glucan, respectively. Besides their role in plant defense, they are known to be involved in diverse physiological and developmental processes, such as embryogenesis, seed germination and flower development, and are also developmentally and hormonally regulated. Initially, in order to study the effects of various cytokinins on chitinase and β-1,3-gIucanase enzyme activities and their gene expression, cotyledons excised from seven-day-old dark-grown cucumber seedlings were treated with water, and cytokinins, viz. benzyladenine, kinetin, zeatin and zeatin riboside. It was observed that chitinase and β-l,3-ghucanase enzyme activities and their transcripts were induced to varying extents following treatments of cotyledons with the cytokinins tested. However, a maximum increase in enzyme activities and their transcript levels was noticed in zeatin-treated cotyledons. Therefore, zeatin was used for further studies. The main objective of the present study was to investigate the cytokinin signal transduction mechanism involving the induction of expression of chitinase and β-1-3-glucanase. In order to obtain insights into the downstream components of the cytokinin-signaling pathway, effects of several agonists and antagonists of the signal transduction components on zeatin-induced chitinase and β-l,3-glucanase activities, and their protein and transcript levels were monitored by enzyme assay, by immunoblot analysis, and by northern analysis, respectively. Treatment of excised dark-grown cucumber cotyledons with staurosporine, a broad spectrum protein kinase inhibitor, reduced the zeatin-induced chitinase and β-l,3-glucanase enzyme activities and the accumulation of their proteins and transcripts. On the other hand, treatment with sodium fluoride, a general inhibitor of protein phosphatases, stimulated the basal chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation, whereas it had no effect on the zeatin-induced enzyme activities and their protein and transcript accumulation. These findings suggested that protein phosphorylation is critical in the cytokinin induction of expression of chitinase and β-l,3-glucanase. Since Ca2+ is known to be an important second messenger in several plant signal transduction pathways, the possible involvement of Ca2+ in the cytokinin-induced expression of chitinase andβ-l,3-glucanase was examined. The results of the present investigation showed that the chitinase and β-1,3-ghicanase activities and their proteins and transcripts were appreciably increased by exogenous CaCl2 treatment in control cotyledons. Treatment of cotyledons with zeatin plus CaCl2 did not result in a further increase in either these enzyme activities or their protein and transcript accumulation as compared to zeatin or CaCl2 treatment alone. The lack of additivity of zeatin plus CaCl2 treatment indicated a common mechanism of action of zeatin and Ca2+ in the induction of these enzyme activities and their gene expression. To test the occurrence of influx of extracellular Ca2+ by cytokinin, cotyledons were treated with the plasma membrane Ca2+ channel blocker, verapamil, and Ca2+ ionophore A23187. Verapamil treatment inhibited the zeatin-induced chitinase and β-1,3-ghicanase enzyme activities and their protein and transcript accumulation. An increase in the intracellular Ca2+ levels by means of Ca2+ ionophore treatment resulted in a significant increase in basal chitinase and β-l,3-glucanase activities and their protein and transcript accumulation. These results suggested that an influx of extracellular Ca2+ leading to increased levels of cytosolic Ca2+ is required for the cytokinin induction of expression of these enzymes. The correlation of chitinase and β-1,3-glucanase enzyme activities and their protein and transcript accumulation in the zeatin-treated cotyledons suggested that the cytokinin zeatin stimulates chitinase and β-l,3-glucanase accumulation at the mRNA level and that the increase in enzyme activities is due to an increase in the amount of the enzyme protein and not by the activation of the existing enzyme. Further, the effect of zeatin on both the enzyme activities and their transcript levels under conditions that inhibit protein synthesis was studied. Treatment of excised dark-grown cucumber cotyledons with cycloheximide, an inhibitor of protein synthesis, in the presence of zeatin, completely nullified the stimulatory effect of zeatin. These results indicated the requirement of cytokinin-induced enhanced concurrent protein synthesis in the observed stimulation of chitinase and β-l,3-glucanase enzyme activities as well as their transcript accumulation Ca2+ In an attempt to isolate the full length cucumber β-l,3-glucanase cDNA from a cucumber cDNA library, we isolated and sequenced one cDNA clone, which was 978 bp long and had a potential polyadenylation signal A ATA A starting 172 bases before the polyadenylation tail A deduced amino acid sequence of the cDNA, which was 242 amino acids in length, apparently encoded a partial β-amyrin synthase. Sequence comparison of the deduced partial amino acid sequence of cucumber β-amyrin synthase with other known plant β-amyrin synthase sequences available in databases revealed significant homologies to β-amyrin synthases from Panax, Pisum and Glycyrrhiza. Southern blot analysis indicated that there was only one β-amyrin synthase gene in the cucumber genome. RT-PCR analysis performed on total RNA isolated from zeatin- and salicylic acid-treated cotyledons using forward and reverse primers designed from the internal regions of the cDNA showed that the transcript levels of β-amyrin synthase were enhanced by both zeatin and salicylic acid. In conclusion, we have demonstrated that chitinase and β-l,3-glucanase accumulation is stimulated by exogenous cytokinin treatment of excised cucumber cotyledons, and this effect is correlated with the content of chitinase and β-1,3-glucanase transcripts as judged by northern analyses. Further, the findings reported in the thesis suggested that Ca2+ influx from extracellular space, protein phosphorylation by staurosporine-sensitive protein kinase(s) and concurrent protein synthesis are required for the signaling of cytokinin-induced expression of both these pathogenesis-related enzymes.

The chlorophyll degradation pathway is central to a number of plant processes including senescence and fruit ripening. However, the regulation of the chlorophyll degradation pathway enzymes is not well understood. The aim of this thesis was to elucidate the genetic mechanisms that control changes in pigment composition leading to fruit flesh yellowing in kiwifruit. Actinidia deliciosa and A. chinensis fruit, which are green and yellow, respectively, provide an opportunity to study the regulation of chlorophyll levels. The expression of genes that code for enzymes of the chlorophyll and cytokinin metabolic pathways was measured using qRT-PCR. Candidates for chlorophyll degradation regulatory points were then characterised for functionality by transient transformation in N. benthamiana. The endogenous cytokinin levels were measured in kiwifruit and transient activation assays were carried out with the promoters of key candidate genes. Overall, expression of the chlorophyll degradation genes was elevated in yellow fruit and expression of biosynthetic genes was higher in green fruit. The chlorophyll degradation-associated protein, STAY-GREEN2 (SGR2), was more highly expressed in yellow fruit, and transient over-expression of SGR was sufficient to drive chlorophyll degradation. Expression of isopentenyl transferase (IPT), the rate-limiting step for cytokinin biosynthesis, showed an increase towards maturity in green fruit, but not in yellow fruit. However, both fruit had similar high levels of cytokinin nucleotides and free bases. A gene coding for O-glucosylation was also highly expressed in green fruit. Green fruit contained higher levels of cytokinin O-glucosides and ribosides towards maturity, suggesting differences in cytokinin signalling, which could lead to regulation of chlorophyll levels via activation of the SGR promoter by transcription factors. It is likely that the chlorophyll degradation pathway and cytokinin metabolism are linked. The differential expression of cytokinin response regulators could lead to differential regulation of cytokinin levels in the fruit of the two species, and possibly differential regulation of the chlorophyll degradation pathway. Progress towards elucidation of the control of chlorophyll levels provides knowledge of this key process in kiwifruit and potentially gene-based markers for breeding new kiwifruit cultivars.

Made available in DSpace on 2014-06-11T19:23:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-22Bitstream added on 2014-06-13T20:50:05Z : No. of bitstreams: 1 marques_ds_me_botib.pdf: 635209 bytes, checksum: 3f687d1811304af12caf18d8de978433 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / 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... / 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)

Developing foliar growth of ivy geraniums (Pelargonium peltatum) bleaches white after exposure to temperatures greater than 30°C. This study investigated chloroplast development in ivy geraniums under heat stress comparing a heat sensitive cultivar, Temprano™ Lavender, and a heat tolerant cultivar, Contessa™ Red. Using transmission electron microscopy and spectrophotometry, foliar bleaching under heat stress was found to be due to an absence of developed chloroplasts within the bleached new growth accompanied by lower chlorophyll content. To determine whether heat stress related foliar bleaching could be prevented, cytokinin and gibberellins were applied in combination, at different rates before, during or after a heat stress event. Applying 50 to 100 ppm gibberellins before heat stress reduced bleaching in new growth. Gibberellins applied at 50 ppm within a week of a heat stress event decreased bleaching. Net photosynthesis and chlorophyll fluorescence was greater in non-heat stressed plants than heat stressed plants.

MECHANISM OF CYTOKININ TRANSPORT ACROSS PLASMA MEMBRANE AND THEIR METABOLISM IN TOBACCO BY-2 CULTURED CELLS Mgr. Petr Klíma / Abstract of Ph.D. Thesis / Prague 2011 Cytokinins (CKs) are plant hormones that play a major role in a number of developmental processes in plants. Those include promotion of cell division, active growth and differentiation, and maintenance of sink-source relationships, as well as control of environmental stress responses. Native CKs are low-molecular derivatives of adenine which seem to act either as paracrine or as long-distance signals. Due to their numerous physiological effects, plants have to precisely control the occurrence of bioactive CK molecules on the levels of the whole plant, its organs, tissues as well as single cells. To achieve this, a concerted action of metabolism and transport processes is required. Studies of the kinetics of CK translocation across plasma membrane in BY-2 suspension-grown tobacco cells suggested the existence of energy-dependent, partially selective transport routes for CK bases and CK ribosides. HPLC analysis of the metabolites of accumulated CKs pointed at their fast degradation or metabolic conversion into physiologically inactive forms. The prevalent ways of inactivation were the degradation to adenine and phosphorylation or phosphoribosyl...

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