No description available.

Light signalling mechanisms are crucial for plant adaptation to environmental changes: it has been found that both light and temperature cues are interpreted by closely interacting signalling pathways. In Arabidopsis thaliana, the seedling hypocotyl has emerged as an ideal model system to study light and temperature control of cell expansion. The light sensitivity is affected by the light intensity (fluence rate), an illustration of which is exemplified in the fluence rate response curve, and also the quality of that light (i.e. the ratio of red: far-red wavelengths of light). Here I have elucidated the phytochrome B (phyB) signalling mechanism in great detail and subsequently created a mathematical model of the red-light signalling pathway. This model is further extended to include changes within the ambient temperature range. Through application of stochastic modelling techniques it was possible to demonstrate the importance of dark reversion within the phyB mechanism in a diurnal cycle. Further, by considering alternative modelling methods and applying a highly integrated mathematical and experimental approach, it has been possible to show how the integration of light and temperature signals drives a molecular switch in transcriptional activity and hence control of cell expansion. This work highlights the importance of a multidisciplinary approach to molecular biology and describes the significance of signal convergence on molecular signalling.

Thesis (Ph. D.)--University of Texas at Austin, 1999. / Vita. Includes bibliographical references (leaves 71-76). Available also in a digital version from Dissertation Abstracts.

Plants modulate their growth and development according to the prevailing light conditions. To detect light signals plants have an array of photoreceptors including the phytochromes which monitor the red and far-red light regions of the light spectrum. Phytochromes regulate gene expression in response to light in part by physically interacting with nuclear-localized bHLH transcription factors called PHYTOCHROME INTERACTING FACTORS (PIFs). PIFs are known to function as negative regulators of photomorphogenesis. Here we show that PIF1, the PIF family member with the highest affinity for phys, is degraded after pulses or continuous red, far-red or blue light in a phytochrome dependent manner. In etiolated seedlings, phyA plays a dominant role in regulating the degradation of PIF1 after a pulse of red, far-red or blue light; while phyB, phyD and other phys also influence PIF1 degradation after prolonged illumination. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the light-induced degradation of PIF1. In response to red, far-red or blue light treatments PIF1 is rapidly phosphorylated, poly-ubiquitinated and degraded via the ubiquitin/26S proteasomal pathway. In addition, we show that PIF1 negatively regulates photomorphogenesis at the seedling stage. The overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12h light/12h dark) blue light conditions. Double mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1 and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis and that light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis. / text

Phytochromes are the photoreceptors mainly responsible for the detection of red and far-red (FR) light and the following responses. HFR1 is a basic helix-loop-helix type putative transcription factor involved in Phytochrome A signaling pathway. First we look at the early phenotype of mutant seedlings lacking a functional HFR1 gene and we show that auxin is involved in the increased hypocotyl phenotype of these seedlings. Northern blots and RT-PCRs showed that ATHB-2, a gene involved in shade avoidance is regulated by HFR1 under FR light. Microarray experiments were performed to find the genes that are early targets of regulation by HFR1.

Characterisation of phytochrome using monoclonal antibodies Mary L. Holdsworth Native oat phytochrome has been purified to homogeneity and used to produce a panel of monoclonal antibodies (mAbs). Selection of mAbs followed early screening against native phytochrome by ELISA, and SDS-denatured phytochrome by "mini" western blotting. Six mAbs which recognised SDS-denatured phytochrome were mapped using proteolytically derived fragments of phytochrome and subsequent immunoblotting. LAS 31 and 33 map to the 6 kDa NH2-terminus and LAS 35 and 41 map to the adjacent 4 kDa sub-NH2- terminal domain. LAS 11 maps to the 64 kDa- chromophore-bearing domain and LAS 32 maps to between 74 and 88 kDa from the NH2-terminus on the COOH- terminal-half of the molecule. A novel protocol for the mapping of conformation-specific mAbs has been developed and used to assign LAS 21, 34 and 42 to the 64 kDa-chromophore-bearing domain. Determination of differential affinities towards Pr and Pfr demonstrated that LAS 42 exhibited a higher affinity for Pfr, LAS 31, 33, 34 and 35 exhibited a higher affinity for Pr. LAS 41 discriminates absolutely against Pfr. LAS 41 has therefore facilitated:- (i) the purification of PfrP, i.e. Pfr which is free of contaminating Pr, (ii) the development of an ELISA for phytochrome photoequilibrium, (iii) the first direct experimental evidence that phytochrome can exist as a stable heterodimer in vitro and (iv) an appraisal of ELISA protocols for determining differential affinities of mAbs towards Pr and Pfr. Spectral analyses of phytochrome in the presence of mAbs have underlined the importance of the 6 kDa NH2-terminus in the maintenance of the spectral integrity of the molecule but have also indicated that the 4 kDa sub-NH2-terminal domain also interacts with the chromophore. Cross reactivity studies amongst phytochrome from monocots and dicots demonstrate that the epitopes recognised by LAS 11, 31, 33, 35 and 41 are not highly conserved. However, LAS 32 recognises phytochrome from every plant species tested, and is therefore recognising a highly conserved region on the molecule.

No description available.

Orientador: Massanori Takaki / Banca: Victor José Mendes Cardoso / Banca: José Eduardo Zaia / Resumo: O objetivo deste trabalho foi conhecer os aspectos envolvidos na germinação de sementes de Tibouchina mutabilis (Vell.) Cogn. (Melastomataceae), espécie característica da encosta úmida da Serra do Mar que ocorre do Rio de Janeiro até Santa Catarina na floresta pluvial da encosta Atlântica. De acordo com a literatura, a espécie é encontrada quase que exclusivamente na mata secundária, onde chega por vezes constituir-se na espécie dominante. Os parâmetros analisados foram: porcentagem final de germinação, velocidade média, o tempo médio e o índice de sincronização e, para maturação, também foi analisada a morfologia do embrião. A faixa ótima de temperatura que proporcionou os melhores valores de porcentagem e velocidade de germinação foi de 25 a 30ºC e na alternância de temperaturas, sob fotoperíodo de 12 horas foi de 20-25°C. As sementes foram sensíveis a luz germinando preferencialmente sob fotoperíodos longos 8 a 12 horas, sendo a quantidade da luz fornecida, determinante na velocidade de germinação, enquanto que a qualidade da luz influenciou as porcentagens finais de germinação, que foi maior nas condições de fotoequilíbrio do fitocromo altos. As sementes embebem no escuro, mas não germinaram na ausência de um período mínimo de 96h de luz. A capacidade germinativa ocorreu com 14 dias pós-antese e a máxima germinação foi atingida aos 21 dias. Observou-se que o melhor indicador de maturação fisiológica para a espécie é o início da abertura natural das cápsulas. Estas características apresentadas indicam que T. mutabilis é uma espécie com características típicas de pioneira / Abstract: The objective of this work was to know the aspects involved in seed germination Tibouchina mutabilis (Vell.) Cogn. (Melastomataceae), feature species of Serra do Mar, occurring from Rio de Janeiro to Santa Catarina, Brazil in Atlantic Forest. The species is found mostly in secondary forest sometimes consisting the dominant species. It was analyzed the final percentage of germination, mean germination rate and daily relative frequency and the synchronization index; It was analyzed the morphology of the embryo and germination capacity during maturation. The range of optimum temperature to germination was of 25 to 30ºC and in the alternating temperature of 20-25°C. The seeds of T. mutabilis were sensitivity to the light, the germination happened preferentially under long photoperiod of 8 to 12h, increase in the light fluence increase the germination rate and increase in the photoequilibrium of phytochrome increase the final percentage germination. The seeds of the T. mutabilis imbibed in darkness, but did not germinate on absence of a minimum period of light 96 hours. The germination capacity appeared in 14 days stage of development, whose embryo in some seeds were partially developed, and the maximum germination capacity was reached in 21 days stage, whose embryo full up the capsule. The best indicator of physiological maturation to this specie is natural open beginning of the capsule. The results showed that T. mutabilis is a pioneer, absolute positive photoblastic specie / Mestre

The ability to withstand environmental temperature variation is essential for plant survival. Formative studies in Arabidopsis have revealed that light signalling pathways has a potentially unique role in shielding plant growth and development from seasonal and daily fluctuations in temperature. In this thesis we further investigate the integration of the light signalling networks and temperature signalling on the molecular level in Arabidopsis. First, we identified the transcript of the bHLH transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1) to be highly dependent on the ambient temperature and under strong control of the red light photoreceptor PHYTOCHROME B (phyB). We found that the long hypocotyl phenotype of the hfr1 mutant was exaggerated in warm conditions, specifically in blue light, downstream of cryptochrome 1. We further show that HFR1 acts in the warm by suppressing the function of PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4, PIF5). PIF4 appears to act as a master regulator of several temperature responses and is directly regulated by the phytochromes. Thus, we define a molecular network where red light and blue light signals together with temperature merge on the regulation of PIF4. In the second part of this thesis, we investigate the relationship between temperature and the fluence rate of light on the inhibition of hypocotyl elongation in Arabidopsis. We find that the response to increasing fluence rates of light is highly dependent on the ambient temperature and that PIF4 and PIF5 acting downstream of the major red light photoreceptor, phyB, are essential for this response. In addition, we provide evidence that in cool conditions, PIF activity is under strong suppression by the gibberellin and HY5 pathways specifically at high fluence rates of red light. The collected work of this thesis highlights the importance of the PIF proteins as integrators of temperature and light signals and furthermore, demonstrates that the response to temperature is highly dependent on both the quality and quantity of light.

Light and temperature are two of the most important non-biological signals that plants must be able to sense and respond to. However, it is not just the individual signals, but the interaction between them that is important. In the dark, Arabidopsis has strongly reduced temperature plasticity. The red light photoreceptor phyB and the PIF family of transcription factors have previously been identified as key regulators of this response. Computer modelling was used to study the interaction between the proteins, to explain counter-intuitive behaviour at certain light conditions. A fluence rate dependent response to temperature was uncovered, where high temperature causes a reversal of the normal seedling response to light. In a PIF and phyB dependent manner, increasing light intensity in the warm can lead to an increase in hypocotyl elongation, the opposite of what has been previously published. This was shown to be dependent on an apically derived response to high fluence rates, independent of the Phytochromes. To extend the analysis beyond the seedling stage, the adult phenotypes of light signalling mutants were subsequently characterised at different temperatures. The temperature dependent control of flowering time and leaf growth were both dependent on Phytochrome and the PIFs, but in apparent opposite directions. This demonstrates that the light signalling system in plants is a complex network of signalling factors, affecting each other in a dynamic process to respond to light and temperature.