Дисертації з теми "Abiotic inducers"

En este trabajo se ha demostrado que el NH4+ actúa como inductor de resistencia frente a estrés salino en cítricos, observándose acumulación de ABA, poliaminas (PAs), H2O2 y prolina. También se ha demostrado que plantas de tomate crecidas con NH4+ muestran una reducción de los síntomas de la enfermedad producida por Pst. El estudio del modo de acción de la NH4+-IR reveló que los mecanismos inducidos en las plantas en respuesta a NH4+, tales como la acumulación de ABA, PAs y H2O2 son clave para la inducción de aclimatación sistémica adquirida (SAA) que confiere a las plantas de tomate resistencia frente a Pst. Por último, se ha demostrado que el virus MNSV induce en plantas de melón una compleja red hormonal de respuesta, así como la acumulación de calosa y ROS. El Hx resultó ser efectivo como inductor de resistencia frente a virus evitando el paso del virus al floema.
In this work, we reveal that NH4+ nutrition in citrange Carrizo plants acts as an inducer of resistance against salinity conditions. We investigated its mode of action and provide evidence that NH4+ confers resistance by priming abscisic acid and polyamines, just as enhancing H2O2 and proline basal content. Moreover, we demonstrated the NH4+ nutrition induces-resistance (NH4+-IR) against Pseudomonas syringae pv tomato DC3000 (Pst) in tomato plants. N-NH4+ plants displayed basal H2O2, abscisic acid (ABA) and putrescine (Put) accumulation. H2O2 accumulation acted as a signal to induce ABA-dependent signalling pathways required to prevent NH4+ toxicity. This acclimatory event provoked an increase of resistance against later pathogen infection. We studied the basal response of melon (Cucumis melo) to Melon necrotic spot virus (MNSV) and demonstrated the efficacy of the hexanoic acid priming that blocks the virus systemic spread. We analyzed callose deposition and ROS production, as well as the hormonal profile and gene expression at the whole-plant level.

>Magister Scientiae - MSc
South Africa is one of two countries responsible for the production of approximately 80% of the world’s Zr. The increase in mining activity has detrimental effects on the environment, especially crop plants, as more pollutants are leached into the soil. Consequently, it is necessary to understand how plants respond to this form of abiotic stress. Therefore, this study focused on determining the physiological and biochemical responses of two genotypes of Brassica napus L (Agamax and Garnet) in response to Zr stress. The levels of cell death, lipid peroxidation and ROS were higher in Garnet, whereas the chlorophyll content was higher in Agamax. Furthermore, native PAGE analysis detected seven SOD isoforms and seven APX isoforms in Agamax, compared to 6 SOD isoforms and 7 APX isoforms in Garnet. The results thus indicate that Agamax is tolerant to Zr-induced stress, whereas Garnet is sensitive. An assay for the rapid quantification of Zr within plant samples was subsequently developed, which revealed that Agamax retained the bulk of the Zr within its roots, whereas Garnet translocated most of the Zr to its leaves. The ability of Agamax to sequester Zr in its roots comes forth as one of the mechanisms which confers greater tolerance to Zr-induced stress. As a consequence, our study sought to use the optical, physical and chemical properties of quantum dots to image the uptake and translocation of Zr in B. napus genotypes. ICPOES was also performed to quantify Zr levels in various plant organs. Data from the ICPOES revealed varying patterns of uptake and translocations between Garnet and Agamax. These patterns were similarly shown in IVIS Lumina images, tracing the transport of QD/Zr conjugates. This method ultimately proved to be successful in tracing the uptake of Zr, and could essentially be a useful tool for targeting and imaging a number of other molecules.

N-nitroso-N-ethyleurea (NEU) and N-nitroso-N-methyleurea (NMU) induced mutants and control plants had been maintained in in-vitro condition for 3 years by continuous sub-culturing and screened 2 generations for resistant mutants selection. In this study highly resistant mutants were regenerated and assessed by leaf discs assay for drought, salt and frost resistance to confirm the persistence of mutation over generations of subculture. Assessment was carried out using mannitol (drought stress), NaCl (salt stress) and freezing (frost stress). Cold-acclimated and non-acclimated leaves were assessed for frost resistance. Results confirmed the persistence of mutations in clones with enhanced tolerance levels to stresses over control plants. Response of individual mutants was different for each of the stresses, some mutants were resistant to two stresses whilst others demonstrated multiple resistance and no one mutant was resistant to a single stress. Acclimation at 4 °C appeared good enough to increase frost resistance compared to non-acclimation. Acclimation also tended to emphasis the difference between mutants and some mutants (K18 & K19) showed highly significant increase in frost resistance at -6 °C compared to control. Responses of in-vitro and in-vivo plants within a clone were correlated. Molecular and biochemical analysis was carried out with objectives (1) To investigate the presence of CBF/DREB1 and COR15 genes in cauliflower (2) To investigate whether the induced resistance can be attributed to the expression of these genes and proline level. The clones (mutants and control) were analyzed under cold acclimation (4 °C) and non-acclimation (22 °C). Total RNA was isolated after 3 h, 6 h, 24 h and 14 d acclimation. Proteins and free proline were isolated after 14 d acclimation. Under non-acclimation, RNA, protein and proline isolated once at end of experiment. cDNA was produced using RT-PCR, with specific primers the gene was detected only in acclimated clones and no PCR product appeared under non-acclimation. The PCR product was isolated, sequenced, and compared the nucleotides and deduced amino acid sequences with other plants. Very high resemblance (- 91 %) with Brassica species (BnCBF5/DREB1, BrDREB1 and BjDREB1B) were found and confirmed the first reporting of the transcription factor BoCBF/DREB1 in cauliflower. This resemblance was reduced to 67% when compared to other plants, confirms that this sequence is conserved in Brassica. The transcript level increased up to 24 h acclimation and then declined. The response of the mutants was different, some showed PCR product at 3 h while others only after 6 h and 24 h acclimation. Through SDS-PAGE and Western blotting, the COR15a protein was detected with specific antibodies obtained from MSU (USA), and the blots appeared in all clones under cold acclimation correlated with frost resistance but under non-acclimation the COR15a constitutively expressed only in 3 mutants with increased frost resistance that confirms the persistence of mutation. The genotypes showed positive correlation between BoCBF/DREB1 expression and frost resistance and this correlation was significant after 24 h and 14 d cold acclimation. The highest R² value was found between BoCBF/DREB1 expression at 14 d and EC% at -6 °C (93.43% of variation accounted for) followed by BoCBF/DREB1 expression at 24 h and EC% at -6 °C (82.57%). The proline level under acclimation increased about 8 times compared to non-acclimation and demonstrated positive and significant correlation with BoCBF/DREB1 expression. Proline also showed positive and significant correlation with frost resistance under cold acclimation but very weak under non-acclimation. The effect of cold acclimation on proline and total protein was evaluated and negative correlation was found to be non significant between free proline and total protein content in clones.

Orientador: Paulo Arruda
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-25T23:29:52Z (GMT). No. of bitstreams: 1 Barreto_PedroPaulo_D.pdf: 2593736 bytes, checksum: 667c2ed03e1e7e51ac393087c3acc7ae (MD5) Previous issue date: 2014
Resumo: A proteína desacopladora mitocondrial 1 (UCP1) é uma proteína mitocondrial codificada pelo núcleo capaz de desacoplar o gradiente eletroquímico usado para a síntese de ATP, dissipando a energia na forma de calor. A descoberta de homólogos e ortólogos da UCP1, sugere outros papéis fisiológicos para estas proteínas. As UCPs podem servir como uma válvula de escape, diminuindo a força protonmotiva (PMF) e reduzindo a produção de ROS em condições desfavoráveis. Plantas superexpressando UCPs se desenvolvem melhor quando submetidas a estresses bióticos e abióticos. Estas plantas demonstraram diminuição na produção de ROS, alteração no estado redox celular, além de um aumento no metabolismo energético e na fotossíntese. Neste trabalho nós investigamos os mecanismos moleculares envolvidos no metabolismo energético celular e resposta a estresses em plantas de tabaco superexpressando a UCP1 de A. thaliana. Demonstramos, através de análises moleculares e genômicas, que a superexpressão da UCP1 é capaz de provocar o aumento na respiração desacoplada em mitocôndrias isoladas, diminuir o conteúdo de ATP intracelular, e desencadear um processo de sinalização retrógrada que resulta na indução de genes mitocondriais e genes responsivos a estresses. Esta sinalização retrógrada resultou na indução do processo de biogênese mitocondrial verificado pelo aumento no número e área mitocondrial por célula, além de alterações morfológicas nestas organelas. O processo de biogênese mitocondrial nestas plantas é acompanhado pelo aumento na expressão de um grande número de genes responsivos a estresses, o que resulta no melhor desempenho e reduzida produção de ROS mitocondrial quando submetidas a estresses abióticos. A análise detalhada do transcriptoma de plantas superexpressando UCP1 em comparação com plantas selvagens demonstrou uma forte conexão entre os metabolismos mitocondrial, citoplasmático e cloroplástico para compensar as alterações provocadas pelo aumento na atividade da UCP1. Um grande número de fatores de transcrição ainda não caracterizados foram identificados e podem representar bons alvos para investigações futuras a respeito da regulação da biogênese mitocondrial e do metabolismo energético em plantas. Os resultados contidos nesta tese nos permitem melhor compreender a flexibilidade do metabolismo energético em plantas e identificar possíveis reguladores do processo de biogênese mitocondrial e resposta a estresses em plantas
Abstract: The mitochondrial uncoupling protein 1 (UCP1) is a nuclear-encoded mitochondrial protein capable of uncouple the electrochemical gradient used for ATP synthesis, dissipating energy as heat. The discovery of UCP1 homologues, and its corresponding orthologues suggest diverse physiological functions for these proteins. UCPs may serve as an escape valve, decreasing the proton motive force (PMF) and preventing ROS production under unfavorable conditions. Plants overexpressing UCPs perform better under biotic and abiotic stresses. These plants show diminished ROS production, alteration of cell redox homeostasis, increased energy metabolism and photosynthesis. In this work we investigated the molecular mechanisms underlying cell energy metabolism and stress response in tobacco plants overexpressing an Arabidopsis thaliana UCP1. We demonstrated through molecular, cellular and genomic tools that UCP1 overexpressing plants is capable of increasing uncoupled respiration of isolated mitochondria, decrease intracellular ATP levels, and trigger a retrograde signaling that resulted in a broad induction of mitochondrial and stress response genes. The retrograde signaling resulted in the induction of mitochondrial biogenesis verified by increased mitochondrial number, area and alterations on mitochondrial morphology. The increased mitochondrial biogenesis in these plants accompanied by the broad increase in the expression of stress responsive genes, may be responsible for the diminished ROS production and the better performance of these plants when submitted to several abiotic stresses. We also performed a detailed analysis of the transcriptome expression of the UCP1 overexpressing plants as compared with the wild type plants. We verified that the UCP1 overexpressing plants exhibited a tight connection between mitochondrial, cytoplasm and chloroplast energy metabolism to accommodate the alterations caused by the increased UCP1 activity. A number of uncharacterized transcription factors seem to be good targets for future investigations on the regulation of plant mitochondrial biogenesis and energy metabolism. The results presented in this work allowed a better understanding of the flexibility of energy metabolism in plants, and the use of this mechanism to identify possible regulators of plant mitochondrial biogenesis and stress response
Doutorado
Bioinformatica
Doutor em Genetica e Biologia Molecular

Tese de mestrado, Ciências do Mar, Universidade de Lisboa, Faculdade de Ciências, 2018
O nosso planeta está a sofrer uma inquestionável mudança climática, cada vez mais acentuada, atribuída a ações antropogénicas. Devido ao constante crescimento populacional e diante dos novos desenvolvimentos tecnológicos tem-se observado uma intensificação de atividades humanas com consequências nocivas para a sustentabilidade do Sistema Terrestre. O aumento contínuo da emissão de gases que contribuem para o efeito de estufa tem como fonte principal a queima de combustíveis fósseis (CO2), a agricultura intensiva (CH4) e o emprego de gases fluorados industriais. O fenómeno do aquecimento global é considerado uma das maiores ameaças ambientais do século XXI. As alterações climáticas, impulsionadas pelas atividades antropogénicas, terão consequências transversais a várias áreas da sociedade particularmente na economia, na saúde e no ambiente, sendo urgente medidas mitigadoras para que estes impactos não se agravem nas próximas décadas. De acordo com os cenários previstos, é muito provável que no futuro para além do aumento da temperatura média global, ocorra um aumento do nível médio do mar e um incremento na frequência e duração de fenómenos climáticos extremos, incluindo secas, cheias, precipitação intensa e ondas de calor. Consequentemente, as alterações climáticas irão afetar profundamente os ecossistemas a nível global, em especial os ecossistemas costeiros. Os Estuários são zonas húmidas costeiras muito ricas do ponto de vista biológico, onde ocorre grande mistura de água doce e água salgada que facilita a acumulação de sedimentos, nomeadamente nas zonas de sapal. As zonas estuarinas, devido à sua localização privilegiada e riqueza biológica, têm atraído, desde sempre, a população humana para as suas margens, servindo de localização para as maiores metrópoles do mundo. As ações antrópicas que, consequentemente, desde há várias décadas, estas regiões têm conhecido, de que são exemplos a exploração excessiva de recursos haliêuticos, o lançamento de resíduos sólidos, a eutrofização causada pela entrada de matéria orgânica, a dragagem e a introdução de espécies invasoras, têm causado impactos negativos. Os sapais têm grande interesse do ponto de vista económico, social e ecológico, prestando serviços de proteção à erosão costeira e à subida do nível do mar, estabilização de sedimentos, habitat para uma diversa variedade de espécies, purificação e retenção de água, remoção e transformação de nutrientes, oportunidades educacionais e recreativas, entre outros, pelo que a sua conservação tem, pois, elevada importância. As plantas de sapal, nomeadamente plantas halófitas, são de um modo geral, tal como o nome indica, extremamente resistentes a elevadas concentrações de sal, e a vários stresses ambientais, tais como contaminação de metais pesados, inundações e eventos térmicos extremos. Contudo, apesar da resiliência demonstrada por estas plantas, as alterações climáticas acopladas com as pressões antrópicas terão um profundo impacto nos sapais costeiros. A resistência e a tolerância das plantas halófitas varia consoante a espécie e o tipo de stress, ou seja, quando expostas a determinados cenários as espécies mais resistentes às novas condições adquirem vantagem em relação às outras, o que poderá alterar a distribuição e frequências das espécies dentro do sapal. No entanto, espécies com uma ampla distribuição geográfica e uma grande capacidade de adaptação e aclimatação revelam, normalmente, variações morfológicas, fenológicas e fisiológicas entre populações, de acordo com os condicionamentos locais. Estas plantas podem apresentar, desta forma, tolerância cruzada o que permite aumentar a resistência a uma gama de diferentes stresses após a exposição a um específico stress. Adicionalmente, as plantas, devido à heterogeneidade ambiental, podem exibir variações intraespecíficas que iram influenciar a maneira como cada população irá responder a futuras alterações climáticas. Deste modo, as consequências diretas das mudanças climáticas podem ter diferentes impactos nas plantas de sapal que apresentam variações intraespecíficas, podendo afetar, de diferente modo, a estrutura e função destas comunidades. Esta tese tem como principais objetivos investigar de que modo, desde logo, o pré-condicionamento abiótico encontrado em diferentes populações de halófitas vai influenciar as suas respostas ecofisiológicas a stresses abióticos, conjeturados pelas previsões locais das alterações climáticas, e, ainda, se estas apresentam uma tolerância cruzada suficientemente significativa de modo a determinar a maneira como o sapal irá evoluir e que consequências ecológicas que poderá sofrer. Mais concretamente, pretendeu-se compreender as consequências das variações intraespecíficas, induzidas por pré-condicionamento, apresentadas pelas populações de duas espécies de halófitas com uma ampla distribuição geográfica, a C3 Halimione portulacoides e a invasora C4 Spartina patens, que colonizam dois sapais vizinhos no estuário do Tejo com diferentes níveis de metais pesados, numa primeira abordagem, face a um aumento de temperatura e numa segunda abordagem, face a um aumento de salinidade. O estudo da influencia da tolerância cruzada ao stress de calor constituiu a primeira abordagem para compreender as variações ecofisiológicas nas respostas das populações pré-condicionadas e não pré-condicionadas a metais pesados de H. portulacoides e S. patens a eventos de calor. Para tal e segundo o “Intergovernmental Panel on Climatic Change”, que indica, com grande certeza, que a frequência e a intensidade dos eventos de calor aumentarão devido ao fenómeno do aquecimento global, as plantas foram expostas a um tratamento de calor durante uma semana e, seguidamente, as suas respostas fisiológicas e bioquímicas foram profundamente analisadas. De acordo com os resultados apresentados na tese, obtidos através de medições fotobiológicas, de atividade enzimática e ocorrência de peroxidação lipídica, acoplando com as analises aos pigmentos e ácidos gordos, a tolerância cruzada indica que o pré-condicionamento a metais pesados influencia significativamente os mecanismos de tolerância ao stress térmico das plantas halófitas. Constatou-se que H. portulacoides pré-condicionado por metais pesados, por tolerância cruzada, apresenta maior resistência ao stress de calor que o H. portulacoides do sapal não contaminado, por outro lado S. patens, quando pré-condicionada por metais pesados, exibe uma pior performance na presença deste stress. Posteriormente estudou-se a influencia do pré-condicionamento a metais pesados na tolerância a aumentos de salinidades em duas populações de H. portulacoides e S. patens. A salinidade devido às alterações climáticas, acabará por crescer em consequência do aumento de eventos de tempestade, de ondas de maré e o do nível médio do mar. Ao contrario da maioria das plantas, as halófitas podem viver e prosperar em solos com salinidades até 855 mM, contudo as espécies halófitas têm respostas dispares à mesma salinidade, abrangendo espécies cujo desempenho óptimo ocorre em ambientes sem sal até espécies onde ambientes altamente salinos são ideais. Sendo assim, indivíduos das populações de H. portulacoides e S. patens dos sapais referidos anteriormente foram expostos a uma variedade de salinidades (0 mM NaCl, 400 mM NaCl, 800 mM NaCl) durante uma semana e logo após o tratamento foram analisadas fisiológica e bioquimicamente. Os resultados, adquiridos através dos mesmos métodos usados no estudo anterior, mostram que o pré-condicionamento de metais pesados tem um papel significativo na forma como as plantas responderam aos stresses. Através disso, verificou-se que S. patens pré-condicionado a metais pesados apresentou um maior fitness aos ambientes salinos, comparando com a população do sapal não contaminado, no entanto H. portulacoides manifestou uma complexa influência não linear à tolerância cruzada, uma vez que exibiu, quando pré-condicionado, uma performance pior quando exposto a 400 mM NaCl enquanto a 800 mM NaCl apresentou um melhor desempenho. Em resumo, é evidente que a halotolerância e a termotolerância das plantas anteriormente estudadas estão claramente interligadas com o seu pré-condicionamento, apresentando uma tolerância cruzada significativa, que vai ter um potencial impacto ecológico e poderá influenciar a forma como dois sapais vizinhos irão evoluir. Assim sendo, assumindo que as condições a que as plantas foram expostas representam as condições que, fruto das alterações climáticas, existirão no futuro, podemos constatar que o sapal contaminado poderá ser mais resistente à invasão por parte do S. patens do que o sapal não-contaminado, quando impactados por eventos de calor e evidencia, também, ser em geral mais resiliente ao aumento de salinidade. Em conclusão, os resultados deste estudo mostram que o pré-condicionamento induz uma complexa variação intraespecífica nos mecanismos de tolerância aos stresses abióticos. Esta variação intraespecífica pode afetar significativamente a maneira como as comunidades de sapais irão evoluir no futuro, podendo influenciar, a estrutura e função de cada sapal. Assim, este estudo contribuiu com informação relevante para o conhecimento de processos ecofisiológicos e pode ser pertinente para modelar e prever possível cenários das mudanças climáticas nos sapais, assim como pode ter interesse para uma gestão de ações de conservação mais eficiente e especificas para cada sapal.
Climate change is having an increasing effect worldwide, causing weighty disturbances in virtually all ecosystems thru arising abiotic variations. Salt marshes ecosystems, although highly resilient, are no exception to the climate shift consequences. Considering halophyte communities resistence and tolerance to abiotic alterations, climate change still makes them vulnerable to dire consequences in their productivity, ending up upsetting the distribution and biodiversity of marsh vegetation, compromising their economic, social and ecological services and unique role as the "Kidneys of the planet". This project aimed to determine heavy metal cross-tolerance thru pre-conditioning to abiotic stresses in halophyte plants. Analyzing the influence that adaptations to local conditions have in tolerance and resistance mechanisms and if this will be relevant in the stress response to the abiotic variations predicted in the future climate change and what could this mean for the shifting ecosystems. For this, it was studied the physiological responses to abiotic stresses in two different populations of the wide spread halophyte species, the native C3 Halimione portulacoides and the invasive C4 Spartina patens, from nearby salt marshes in the Tagus estuary, with the major difference between them being heavy metal soil content. Firstly, on the basis that the frequency and intensity of warming events will rise, in duration and frequency, due to global warming, the halophyte plant groups where exposed to a warming event and then analyzed photobiologically and biochemically. The cross-tolerance results shown that heavy metal pre-condition enhanced the heat resistance mechanisms in H. portulacoides and worsened S. patens ability to cope with these changes, when compared to their non-pre-conditioned counterparts. This can have important consequences in the way the non-contaminated and heavy metal contaminated salt marshes will react to environmental changes, that can lead to possibly different ecological consequences. Considering the predicted climate change scenarios, it can be claimed that the contaminated marsh can be more resistant to invasion than the pristine marshes, were the warming events will benefit the exotic S. patens in relation to the native H. portulacoides. Secondly and taking in to consideration that land salinization is an increasing problem around the world and climate change will intensify this phenomenon especially in coastal lands, due to tidal surges, storm surges and sea level rise coupled with the increasing evaporation because of global warming, the halophyte species from both the studied salt marshes were exposed to a variety of salt treatments. When analyzed, the results exposed a noteworthy influence that the heavy metal pre-conditioned has in the salinity stress responses. In view of the future changes, it can be argued that heavy metal contaminated marshes are less resistant to the S. patens increasing colonization when exposed intermediate salt concentrations and in high salinities both H. portulacoides and S. patens have overall better salinity tolerance than their counterparts from the non-contaminated marsh. Concluding, this work shows that the heavy metal pre-conditioning has a complex and considerable influence in the plants tolerance and resistance mechanisms to several abiotic stresses, presenting an intraspecific physiological variation that can impact how the halophyte communities will respond to the upcoming environmental changes. According to our research it can be said that the heavy metal contaminated salt marsh will present significantly different ecological damages than their neighboring salt marsh.

碩士
臺灣大學
植物科學研究所
95
Heat stress is one of the major environmental stresses. When plants are exposed in the elevated temperature, a general physiological process called heat shock (HS) response makes plants accumulate amounts of proteins called heat shock proteins (HSPs) to survive from heat stress. Among HSPs, small HSPs (sHSP) represent the unusual abundance and complexity in plants, and are mainly regulated by heat shock factors (HSFs). There are 21 HSFs in Arabidopsis in compared with 4 in mammalian, but the roles of each HSF in controlling sHSP gene expression and thermotolerance remain largely unaddressed. Hence, this study used reverse genetics analysis to clarify the roles of these HSFs in Arabidopsis. We screened 11 AtHSFs T-DNA insertion lines and 8 AtHSFs null mutant lines including AthsfA1e, AthsfA2, AthsfA4c, AthsfA5, AthsfA6a, AthsfA7a, AthsfA7b, and AthsfB2b were characterized and confirmed by RT-PCR. Through microarray database mining and RT-PCR analysis, some HSFs were induced by certain abiotic stresses. When HSF knock-out plants were treated with various abiotic stresses to examine sHSPs expression, the experiment results confirmed that under heavy metals treatments several sHSPs expression in AthsfA7a mutants was affected, and in AthsfA6a mutant sHSPs expression were also interfered during salt and osmotic stresses. Hence, AtHSFA7a and AtHSFA6a may regulate sHSPs expressions respectively in response to heavy metal and osmotic or salt stresses. When testing the thermotolerance in each HSF null mutant plants, results showed they didn’t lose the basal and acquired thermotolerance, except in AthsfA2. In summary, the expression profile of sHSPs and HSFs under HS and other abiotic stress as well as the regulation of sHSPs expression by several HSFs are established. In addition, AtHSFA7a and AtHSFA6a specifically functioning in regulation of sHSPs expressions in response to heavy metal and osmotic or salt stresses are suggested.

碩士
國立臺灣大學
微生物與生化學研究所
96
There are about 1000 E3 ligases in Arabidopsis, which recognize specific protein substrate and catalyze the ubiquitination reaction to these proteins. The recognition between E3 ligases and their substrate proteins occurs in response to many plant signaling pathways directly or indirectly. Previous yeast two-hybrid experiments indicated that AtMAPR5 might interact with a RING protein, RING3g, which is encoded by At3g58030. In vitro ubiquitination assay was established to find out whether AtMAPR5 is the substrate protein of RING3g. RING3g and RING2g (encoded by At2g42030), a close homolog of RING3g, showed the E3 ligase activity in the in vitro ubiquitination assay. However, RING3g did not show the ability to modify AtMAPR5 in vitro and might not be the E3 ligase of AtMAPR5. To expand our studies to find out novel E3 ligases involved in the abiotic stress signaling, eight RING proteins (R1 ~ R8) were cloned based on microarray database. Seven recombinant proteins among them possessed the activity of autoubiquination using in vitro ubiquitination assay. The physiological roles of the RING proteins still await to determine. Finally, the possibility of how to employ in vitro ubiquitination assay in the discovery of novel RING-type E3 ligases involved in salt stress signaling is also discussed.

碩士
國立臺灣大學
農藝學研究所
96
Through cold acclimation, plant can increase its tolerance capability upon exposure to low temperature. Previous study showed that CBFs/DREBs (C-repeat binding factor/ dehydration response element binding factor) were the major transcription factors that involved in cold acclimation process. The function of CBFs/DREBs evolved highly conserve between dicot and monocot. Recent study from Arabidopsis has defined ICE1 (inducer of CBF expression), a bHLH (basic helix-loop-helix) protein, as an important transcriptional factor that acts on the promoter of CBF gene and regulates its expression. In this study, based on rice functional genomic approach with transgenic rice analysis, we aimed to understand physiological function of Arabidopsis AtICE1 and Barly HvICE1 under different abiotic stresses. Meanwhile, the action mold of AtICE1 and HvICE1 will be compared under various stresses. To reach this goal, first, by bioinformatics search at least four of OsICE genes were found in rice genome. From currently available rice microarray data revealed that OsICE1 expression was highly induced by salt and drought but not affected in low temperature. OsICE2 and OsICE3 transcripts were repressed upon exposure to salt and drought environment. On the other hand, OsICE4 expression was salt and drought induced. Besides, OsICE3 and OsICE4 gene expression were both increased under low temperature stress. Then, we used TNG67 (Oryza sativa L., japonica; cold and salt tolerant but drought sensitive) and TCN1 (Oryza sativa L., indica; cold and salt sensitive but drought resistant) rice cultivars to investigate OsICEs; OsDREBs and OsDREBs regulon-related downstream genes expression profiles under abiotic stress treatments. The results indicated that OsICE2 expression level were down-regulated quickly in TCN1 at low temperature. And the amount of OsDREB1F、OsDREB1G、OsDREB1H、OsDREB1I and OsDREB1J expressions in TCN1 were also less than those of TNG67. To further elucidate the physiological effects of AtICE1 and HvICE1 under various abiotic stresses, we generated ICEs-overexpressed transgenic rice lines, 35S::AtICE1 and 35S::HvICE1. By Southern blotting analysis, TAIL-PCR, and PCR-based genotyping, we determined the copy numbers of transgene, T-DNA inserted flanking sequence and obtained either one or two copies of homozygous transgenic lines. RT-PCR result showed under normal growth condition indeed we can detect the overexpression of ICE genes in 35S::AtICE1 and 35S:: HvICE1 transgenic rices. However; compared to low-temperature stress-treated wild type plant, the whole gene expression profile of ICE-corresponding downstream genes (OsDREBs) did not obviously changed. The physiological analysis of abiotic stress tolerance assay, including chlorophyll, malondialdehyde (MDA) and proline content measurements showed that 35S::AtICE1 transgene rice with OsDREB1A; 1B; 1C and 2B transcripts enhanced could increase cold tolerance but not for drought and salt tolerance. 35S::HvICE1 transgene rice that with slightly OsDREB 1B; 1C and 1E gene expression increased could raise up its drought and cold tolerance but not salt tolerance. Taken together, the above results suggested that AtICE1 and HvICE1 may function not exactly the same in cold acclimation pathway. This may due to other ICEs co-operate in the regulation of CBFs/DREBs and CBFs/DREBs regulon-related gene expression or ICEs activity can be adjusted through post-translation modifications that lead to different responses when exposure to different abiotic stresses.

碩士
國立臺灣大學
植物科學研究所
100
Plants face various environmental stresses, such as drought, salinity, heat and cold directly. In response to heat stress (HS), heat shock response (HSR) is a conserved mechanism of increasing heat shock protein (HSP) genes expression through a heat shock factors (HSFs)-dependent mechanism. According to microarray databases, AtHSFA6a is induced by salt and osmotic stress, while AtHSFA6b expression is responsive to cold, salt, osmotic and drought treatment; in addition both genes are upregulated by ABA treatment suggesting the AtHSFA6s may involve in ABA-mediated responses. This report describes our investigation of the role of AtHSFA6s in response to abiotic stress in Arabidopsis by genetic and molecular approaches. Both AtHSFA6s were induced by abscisic acid (ABA), salt and osmotic stress. Downregulated AtHSFA6b expression in ABA-deficient and ABA-insensitive mutants indicates that ABA signaling is required for AtHSFA6b expression. Notably, ABRE acts as the functional cis-element in the promoter region of AtHSFA6b, suggests AtHSFA6b is a member involved in ABA-dependent pathway. Analysis of cotyledon greening, root growth and salt tolerance in mutant lines and transgenic plants indicated that AtHSFA6b participates in ABA-mediated stress responses and acts as a positive regulator. In addition, thermotolerance tests also showed AtHSFA6b is involved in basal- and acquired-thermotolerance. These studies reveal AtHSFA6b is a positive regulator in ABA-mediated stresses, and add a new signaling pathway into the complex HSF network.

Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, South Africa
In the forestry industry the requirement for the maintenance of a broad genetic base is integral to the success of breeding programmes such as those for Eucalyptus grandis, an important species to the South African forestry industry. Plant cryopreservation is an economical option to maintain such a genetic base, as it allows storage of vegetative materials at sub-zero temperatures, while maintaining juvenility. However, successful cryopreservation of this sub-tropical species has been restricted by its sensitivity to cryopreparative drying. As a consequence, the viability of material is reduced even before reaching the freezing stage. Despite this abiotic stress restriction, evidence of upstream ‘cross-talk’ implying downstream ‘cross-tolerance’ has suggested the possibility that cold acclimation may improve the tolerance responses towards dehydration stress by means of ‘cross-acclimation’. It was therefore the aim of the study to understand some of the physiological and biochemical responses of in vitro E. grandis shoots to different non-freezing low (chilling) temperatures and exposure periods, and to establish an appropriate ‘cross-acclimation’ regime for the physical drying pre-treatment. E. grandis shoot clusters (4-8 leaves and 2-5 axillary buds) were exposed to the chilling temperatures of 5°C, 10°C or 15°C for 1 or 3 days. The physiological and biochemical responses were evaluated, and thereafter the appropriate cold acclimation (or ‘cross-acclimation’) regime selected. The appropriate physical drying time was also selected for shoot clusters according to their physiological responses. When the appropriate regimes had been determined, the physiological and biochemical responses of shoot clusters treated consecutively to cold acclimation and then physical drying were evaluated. The physiological responses evaluated were water content, viability, and vigour (i.e. the number of visible axillary buds and shoots produced over 2 weeks). The biochemical responses measured were the concentrations of: 1) total soluble sugars, 2) starch, 3) phenolic acid, and 4) superoxide. The data suggested that the appropriate cold acclimation regime was treatment at 10°C for 3 days. This was based on the accumulation of the high levels of phenolic acid (3.05 ± 0.09 mg GAE.g-1 FWS) and positive vigour responses (11.90 ± 0.60 visible axillary buds/week and 3.10 ± 0.20 visible shoots/week), compared with the other chilling temperature treatments. The appropriate drying time selected for shoot clusters was 80 min over activated silica gel to achieve a water content of 0.32 ± 0.04 g water.g-1 FWS. In the dried material there were high levels of soluble sugars (47.65 ± 1.90% of the fresh weight of shoots) and unknown components that accounted for 48.10 ± 1.86% of the fresh weight, followed by phenolic acid (3.09 ± 0.05%) and proline (0.490 ± 0.011%). Despite these measured responses, viability of the shoots was impacted by drying, dropping to 88.9 ± 3.9%. When shoot clusters were pre-treated at 10°C for 3 days and then physically dried, viability of all (100%) the material was retained and the water content did not drop as low as with physical drying alone, dropping to 0.52 ± 0.05 g water.g-1 FWS. The biochemical responses showed that tolerance was strongly dependent on a high proportion of soluble sugars (83.66 ± 1.48% of the fresh weight of shoots) and phenolic acid (3.77 ± 0.12%), followed by proline (0.406 ± 0.018%). The study had confirmed that ‘cross-acclimation’ through means of cold acclimation (chilling pre-treatment at 10°C for 3 days) can induce ‘cross-tolerance’ towards physical drying, where osmotic adjustments and osmoprotection appeared to have been improved. It is therefore possible that this may have the potential to improve survival during the latter stages of the cryopreservation procedure, despite the higher retention of water in shoot clusters after drying.