The constitutively obsereved up-regulation of Hsp70 expression often led to premature conclusions about its critical role as a repair mechanism of cold injury that is, besides, expressed by protein misfolding/denaturation. In this study, we analyze the cold tolerance and the expression of 24 different mRNA transcripts of Hsp complex and other genes, that are associated with the repair of injury caused by cold. We use two strains of D. melanogaster: the wild type and the mutant type Hsp70- null, that lacks all 6 copies of the gene hsp70. We found out, that the larvae of two strains do not differ in their patterns of target genes expression during long term acclimation nor during recovery from chronic cold exposure and acute cold shock, therefore there is no transcriptional compensation of any other Hsp gene for the missing hsp70 in Hsp70- strain. The cold tolerance of Hsp70- strain larvae was impaired only, when they were exposed to strong acute cold shock by temperatures below -8°C. No difference in cold tolerance was observed, when the larvae were exposed to chronic cold exposure in 0°C or to mild acute cold shock by temperatures up to -4°C. Based on our results we assess, that the cold injury caused by strong acute cold schock is of another nature than caused by mild cold conditions and only in the first case Hsp70 expression is critical for the repair of cold injury in Drosophila melanogaster larvae.

A obtenção de cultivares de arroz de alta produtividade e com tolerância a baixas temperaturas para a zona temperada da América Latina é um dos mais importantes desafios para a agricultura local. Neste trabalho buscou-se estimar parâmetros genéticos visando o entendimento do controle genético de tais caracteres para fins de melhoramento genético. Foram avaliadas 13 linhagens de arroz, separados em dois grupos: Grupo 1 composto de seis linhagens suscetíveis ao frio, e Grupo 2 composto de sete linhagens tolerantes ao frio. Os dois grupos foram cruzados de acordo com o delineamento em dialelo parcial (MIRANDA FILHO; GERALDI, 1984) originando 42 combinações híbridas. Os 55 tratamentos (42 híbridos e 13 genitores) foram avaliados no campo em 2005, em duas épocas de plantio, no Centro Internacional de Agricultura Tropical - CIAT. Utilizou-se um delineamento em blocos casualizados com quatro repetições e parcelas constituídas de uma linha com 17 plantas. Os mesmos tratamentos foram avaliados para tolerância ao frio em câmara de crescimento, em três experimentos, utilizando solo como substrato, temperaturas de 16 °C e 12 °C durante o dia e noite, respectivamente, e fotoperíodo de 12 horas com luz artificial. Utilizou-se o mesmo delineamento experimental, com três repetições e parcelas constituídas de uma linha com 20 sementes. Os resultados mostraram a existência de heterose para todos os caracteres agronômicos (altura da planta, número de perfilhos, comprimento da panícula, número de grãos por panícula, porcentagem de esterilidade e peso de 1.000 grãos) e de tolerância ao frio (índice de emergência e produção de matéria seca). O sentido da dominância foi negativo para dias para florescimento e positivo para os demais caracteres, com contribuições desiguais dos genitores para a heterose média. Os efeitos da capacidade geral de combinação (CGC) e da capacidade específica de combinação (CEC) foram importantes para todos os caracteres; entretanto, a CGC foi mais importante que a CEC. Os dois grupos de genitores têm alelos favoráveis para os caracteres agronômicos e para a tolerância. Algumas linhagens como Irga 417 e FL03188 do Grupo 1, FL04423, FL04402, e FL04452 do Grupo 2 apresentaram os menores índices de esterilidade nos cruzamentos intra-grupos. Os genótipos CT6748, Quilla 173201 e Quilla 145601 foram os mais tolerantes a baixas temperaturas e, portanto, promissores para serem utilizados em programas de melhoramento genético de arroz para condições de baixas temperaturas. / The development of high yielding and cold tolerant rice cultivars for the temperate region of Latin America is one of the most important challenges for the local agriculture. The objective of the present work was to estimate the genetic and phenotypic parameters related to the control of cold tolerance and agronomic traits, in order to obtain a better understanding of the genetic control of these traits for breeding purposes. The genetic material comprised two sets of rice lines: Group 1 composed by six cold susceptible lines and Group 2 composed by seven cold tolerant lines. The two groups were crossed according a partial diallel design (MIRANDA FILHO; GERALDI, 1984) giving rise to 42 hybrid combinations. The 55 entries (42 crosses plus 13 parents) were evaluated in 2005 under field conditions, at the International Center for Tropical Agriculture - CIAT, in a randomized complete block design with four replicates, in two planting dates; plots were single-rows with 17 plants. The same entries were evaluated for cold tolerance in growth chambers, in three experiments, using soil as substrate, temperature of 16 °C and 12 °C during the day and night, respectively, and photoperiod of 12 hours using artificial lights. The experimental design was the same with three replications per experiment; and plots were single-rows with 20 seeds. General results revealed the presence of heterosis for the agronomic traits (plant height, tiller number, days to flowering, panicle length, grains by panicle, sterility and one-thousand grain weight) as well as for cold tolerance traits (emergence index and dry matter production), with different contributions of the parents to the mean heterosis effect. The direction of the dominance was negative for days to flowering and positive for all others traits. General combining ability (GCA) and specific combining ability (SCA) were important for all traits; however, the GCA was more important than SCA. Both groups showed the presence of favorable alleles for agronomic and cold tolerance traits. Several lines such as Irga 417 and FL03188 from the Group 1, FL04423, FL04402, and FL04452 from the Group 2, presented lower sterility in the inter-group crosses. The tolerant lines, CT6748, Quilla 173201 and Quilla 145601 were considered the best parents to be used as cold tolerant donors in rice breeding programs.

Made available in DSpace on 2014-08-20T13:44:38Z (GMT). No. of bitstreams: 1 dissertacao_democrito_amorim_chiesa_freitas.pdf: 403064 bytes, checksum: 624a0297ee84d15e4d39c65e50fc1b9c (MD5) Previous issue date: 2005-08-31 / To evaluate the performance of a thermogradient table in the maintenance of different temperatures and ranking genotypes of rice according to its germination capacity at law temperature. For this procedure it was used a thermogradient table of the Seed Analysis Laboratory of Faculdade de Agronomia Eliseu Maciel of Universidade Federal de Pelotas RS, manufactured in Brazil. Forty genotypes of rice were used, ranking through the determination of the percentage and speed of the germination in four temperatures (10ºC, 13ºC, 16ºC and 19ºC) and the behavior of the genotypes in relation to the stress at low temperature. It was concluded that: a) there is great variability among genotypes of rice, as for the tolerance of the germination in low temperature, with emphasis in the following varieties: Colonial, IRGA 418, BR IRGA 411 and EEA 406. b) There are genotypes of rice that present more than 50% of germination in temperature of 10ºC up to two weeks after the sowing. c) The thermogradient table is an equipment capable of maintaining stable temperature in each ruler, with a maximum variation of one degree Celsius. d) The thermogradient table allows to manitor the performance of seeds of genotypes of rice, in different temperatures, simultaneously / Para avaliar o desempenho de uma mesa termogradiente na manutenção de diferentes temperaturas e ranquear genótipos de arroz quanto à capacidade de germinação à baixa temperatura, foi empregada uma mesa termogradiente do Laboratório de Análise de Sementes da Faculdade de Agronomia Eliseu Maciel da Universidade Federal de Pelotas RS, fabricada no Brasil. Foram utilizados quarenta genótipos de arroz, com ranqueamento através da determinação da percentagem e velocidade da germinação em quatro temperaturas (10º, 13º, 16º e 19ºC) e o desempenho dos genótipos em relação ao estresse à baixa temperatura. Concluiu-se que: a) Há grande variabilidade entre genótipos de arroz, quanto à tolerância a germinação em baixa temperatura, destacando-se o Colonial, IRGA 418, BR IRGA 411 e o EEA 406. b) Determinados genótipos de arroz apresentam germinação superior a 50% em temperatura de 10ºC, até duas semanas após a semeadura. c) A mesa termogradiente é um equipamento capaz de manter estável a temperatura em cada régua, com variação máxima de um grau Celsius. d) A mesa termogradiente permite monitorar o desempenho de sementes de genótipos de arroz, em diferentes temperaturas, simultaneamente

Made available in DSpace on 2014-08-20T13:44:44Z (GMT). No. of bitstreams: 1 tese_democrito_amorim_chiesa_freitas.pdf: 404655 bytes, checksum: c873f95da6df2da75ff9fd90ecb3dc2b (MD5) Previous issue date: 2009-03-25 / The need to widen the seeding window of irrigated rice cultivars capable of germinating and producing normal seedlings under low soil temperatures led to the execution of this work, at the laboratory of the Plant Science Department from the Faculty of Agronomy Eliseu Maciel , Federal University of Pelotas and greenhouses at Embrapa s Temperate Climate (CPACT - Pelotas/RS) and Rice & Bean (CNPAF - Goiânia/GO) Experimental Stations. The experimental work focused on the identification of irrigated rice genotypes from an F2 generation tolerant to low soil temperatures at the germination and emergence stages, through the selection of cDNA fragments differentially expressed as the result to low temperature exposure during the germination phase. The procedure consisted of three stages; First stage involved selection, through physiological traits associated to genotype tolerance and sensitivity to germination onset and emergence under low temperature, among the 104 genotypes tested. The Second stage focused on the screening for variability for low temperature tolerance trait among the F2 progeny of a dialellic set of crossings, while the Third stage related to differentially expressed cDNA fragment selection, among embryos from low temperature tolerant and sensitive genotypes. Main results are as follows; a) A large variability for traits expressing tolerance to germination and emergence under low temperatures was determined among the genotypes tested, with BRS Atalanta, LTB 06002, LTB 06012 and LTB 06020 genotypes exhibiting superior germination values, whereas genotypes LTB 06014, Nourim Mochi and Oro exhibited higher emergence values. b) The rice genotypes with high germination values under low temperature conditions did not show the same trend during the emergence stage. c) The cDNA/AFLP technique proved effective in producing and identifying rice gene fragments differentially expressed, under ambient conditions of low temperature during germination. d) Four polymorphic fragments were identified for genotype BRS Firmeza at a temperature of 13°C, thus indicating that four genes were expressed under this condition. / Buscando ampliar a janela de semeadura, com cultivares de arroz irrigado que germinem e desenvolvam plântulas normais em baixa temperatura de solo, realizou-se este trabalho nos laboratórios do Departamento de Fitotecnia da Faculdade de Agronomia Eliseu Maciel da Universidade Federal de Pelotas e nas casas de vegetação da Embrapa Clima Temperado - CPACT (Pelotas/RS) e Embrapa Arroz e Feijão CNPAF (Goiânia/GO). O estudo objetivou identificar genótipos de arroz irrigado tolerantes a baixa temperatura, nos estádios de germinação e emergência, o caráter germinação no frio, na população F2 e selecionar fragmentos de cDNA diferencialmente expressos no frio na germinação. O trabalho foi dividido em três etapas: Primeira etapa, seleção fisiológica, dos genótipos tolerantes e sensíveis ao frio na germinação e na emergência, entre os 104 genótipos estudados, em temperaturas previamente selecionadas; Segunda etapa, observação da existência de variação, na descendência dos cruzamentos dialélicos, para a tolerância ao frio na população F2 , em relação aos pais; Terceira etapa, seleção dos fragmentos de cDNA diferencialmente expressos, entre os embriões de genótipos tolerante e sensível ao estresse abiótico do frio. a) Há variabilidade, no arroz irrigado, quanto à tolerância à baixa temperatura no estádio de germinação e emergência, destacando-se na germinação o BRS Atalanta, LTB 06002, LTB 06012 e LTB 06020, na emergência o LTB 06014, Nourim Mochi e Oro. b) Os genótipos que apresentaram melhor germinação, em baixa temperatura, não mantiveram a superioridade na emergência. c) A técnica de cDNA/AFLP é eficiente para produzir e identificar fragmentos de genes diferencialmente expressos, em genótipos de arroz irrigado durante a germinação, sob estresse de baixas temperaturas. d) No genótipo BRS Firmeza, foram identificados quatro fragmentos polimórficos na temperatura à 13ºC, indicando que quatro genes se expressaram nesta temperatura.

Abstract The life histories of ectoparasites are shaped by both host and off-host environment. A suitable host is primarily needed during reproduction, whereas juvenile stages outside the host are directly exposed to environmental variability. Viviparity, i.e. the development of an embryo inside the body of the mother resulting in large offspring size, increases offspring survival. The production of large offspring has its consequences in terms of high variation in offspring age and in the environment that each young individual will face. I used a viviparous ectoparasite, the deer ked (Lipoptena cervi), to investigate the consequences of long reproductive lifespan and varying offspring environment on offspring life-histories and seasonal adaptations. Offspring life-histories varied seasonally. I showed that the resources provided by the deer ked females determine offspring performance throughout its off-host period. Offspring size increased towards the spring and the end of the reproductive period, and simultaneously offspring survival and cold tolerance increased. Seasonal variation in offspring size did not reflect the resources that would guarantee offspring survival during the longest diapause or the highest cold tolerance during the harshest winter period. Diapause intensity varies with birth time according to the expected length of the winter ahead. However, the deer ked pupae, regardless of their age, overwinter at an opportunistic diapause, which may be terminated rapidly only by an exposure to high temperature. Contrary to general observations, photoperiod has no role in regulating the seasonal shifts of the deer ked. Neither is high cold tolerance associated only with diapause, but it remains high through four seasons, also in the active developmental and adult stages. I also evaluated the effects of life-history variation on the invasion potential of the deer ked. I conducted a large-scale transplant experiment to test the survival and pupal development at and beyond the current range. I found that the lower spring and summer temperatures and the shorter growth season in the north cause a deterioration in pupal performance and shorten the flight period. However, the colder climate may not totally prevent further spread. A more important factor that will affect deer ked invasion is host availability, and especially in Finland, the density of the moose population. Seasonal variation in offspring life histories in viviparous ectoparasites differs from the variation patterns reported in most invertebrates. This may be due to the extremely large offspring size and to the fact that maternally derived resources determine offspring performance through the entire off-host period. Variation in offspring performance is thus determined by maternal resources and seasonal variation in the condition of the moose. / Tiivistelmä Useimpien ulkoloisten elinkierto on riippuvainen isännästä ja isännän ulkopuolella kasvavien jälkeläisten kohtaamista ympäristöoloista. Viviparia eli jälkeläisen kehitys naaraan sisällä ja siitä usein seuraava suuri jälkeläiskoko parantavat jälkeläisten selviytymistä. Suurten jälkeläisten tuottaminen pitkällä aikavälillä johtaa siihen, että eri-ikäiset jälkeläiset kohtaavat vuodenajasta riippuen hyvin erilaiset olosuhteet. Väitöstyössäni tarkastelin Suomessa nopeasti yleistyneen hirvieläinten ulkoloisen, hirvikärpäsen (Lipoptena cervi), avulla, mitä seurauksia viviparialla, pitkällä lisääntymiskaudella ja ympäristön vuodenaikaisvaihtelulla on jälkeläisten elinkierto-ominaisuuksiin. Väitöskirjatyössäni havaitsin, että hirvikärpäsjälkeläisten elinkiertopiirteet vaihtelevat jälkeläisen syntymäajan mukaan. Osoitin myös, että hirvikärpäsnaaraan jälkeläisilleen tarjoamat ravintovarat määrittelevät jälkeläisten isännästä riippumattoman elinkierron aikaisen menestyksen. Jälkeläisten keskimääräinen koko kasvoi lisääntymiskauden edetessä talvesta kohti kevättä, jolloin myös selviytyminen ja kylmänsietokyky paranivat. Jälkeläiskoon vuodenaikaisvaihtelu ei näin ollen vastaa jälkeläisten tarvitsemia resursseja suhteessa talvehtimisen pituuteen eikä korreloi koville talvipakkasille altistumisen todennäköisyyden kanssa. Lepotilan keston määrittelevä diapaussin syvyys vaihteli syntymävuodenaikaa vastaavasti. Diapaussin havaitsin kuitenkin olevan ensisijaisesti opportunistinen, jolloin pelkästään korkea lämpötila voi nopeasti päättää lepotilan kaikenikäisillä yksilöillä. Vastoin yleisiä käsityksiä valorytmi ei vaikuta diapaussin säätelyyn. Vastoin ennakko-odotuksia kylmänsietokyky säilyy korkeana vuoden ympäri ja kaikissa tutkituissa elinkierron vaiheissa. Sovelsin tutkimieni elinkiertopiirteiden vaikutusta myös lajin invaasiokykyyn, ja tutkin istutuskokeen avulla koteloiden selviytymistä ja kehitystä nykyisellä esiintymisalueella ja sen pohjoispuolella. Alhaisemmat kevät- ja kesälämpötilat sekä lyhyempi kasvukausi vähentävät aikuiseksi selviytymistä ja lyhentävät lentoaikaa syksyllä. Ilmastotekijöiden suhteen hirvikärpänen voisi esiintyä nykyistä pohjoisempana. Tärkein tekijä hirvikärpäsen leviämistä tarkasteltaessa on kuitenkin sopivien isäntäeläimien saatavuus ja Suomen oloissa erityisesti hirven eli hirvikärpäsen pääisännän kannan tiheys. Tutkimukseni perusteella ulkoloisten vivipariasta seuraava jälkeläisten elinkiertopiirteiden ajallinen vaihtelu eroaa muiden selkärangattomien vastaavasta vaihtelusta. Yhtenä syynä eroihin lienee se, että hirvikärpäsnaaras tuottaa erityisen suuria jälkeläisiä ja että jälkeläiset puolestaan ovat täysin riippuvaisia emon antamista resursseista. Emon lisääntymisresurssit ja hirven kunnon vuodenaikaisvaihtelu vaikuttavat mahdollisesti siihen, minkälaisia jälkeläisiä hirvikärpänen milloinkin kykenee tuottamaan.

<p>As the world population continues to grow, it will be challenging to manage resources, reduce environmental pollution and maintain growing demand for food production. Controlled environment agriculture (CEA) is a novel solution to reduce freshwater use in agriculture, minimize environmental pollution from agriculture sector, and meet the growing food demand. CEA allows for the year-round cultivation in inhospitable climatic conditions. Hydroponics is a common method of growing crops in CEA, where plants grow in a solution enriched with nutrients and oxygen. The technique significantly reduces water use and fertilizer run-off during production. In the United States, lettuce is one of the most important crops grown using hydroponics.</p> <p> Hydroponic production uses several methods to grow lettuce including nutrient film technique (NFT) and constant flood table (CFT). Moreover, several cultivars of lettuce are grown in the Midwest. There is a lack of knowledge on whether optimal fertilizer concentrations change depending on the cultivar or hydroponic production system. Little information is known about the suitability of a cultivar to a specific method of hydroponic production. For year-round lettuce production in hydroponics, supplemental lighting (SL) and heating are required in the Midwestern regions of the U.S. The energy requirements for SL and heating can be too costly in winter for some growers to produce crop year-round. In addition to light quantity, spectral composition of light can impact growth. Heating the root zone to produce a micro-climate may be more efficient than heating the entire greenhouse and possibly reduce overall heating costs. However, information on spectral composition of light and the efficacy of root zone heating is unclear, at best. Certain cultivars that can tolerate cold stress can be more suitable in the U.S. Midwest during winter. Lettuce cultivar screening for yield under cooler environments is limited. </p> <p> A completely customizable hydroponic production system that can aid in conducting research related to above-mentioned issues was built as a part of my Master of Science program. Using this system, 24 popular cultivars from four lettuce groups were evaluated for productivity during summer/fall under different concentrations of fertilizer solution, and in two production methods including NFT and CFT during spring. In addition, yield of all 24 cultivars were evaluated under 10, 15.5 and 21.1 °C in a growth chamber. The eight best performing cultivars from the summer/fall trial were evaluated during the winter in a greenhouse with the addition of SL and root zone heating with minimal ambient air heating. </p> <p> Results indicated that the lowest level of electrical conductivity (EC) of the fertilizer solution used (1.3 dS·m^-1) resulted in highest yield, regardless of cultivar or method of production. Among the 24 cultivars; Red Sails (Leaf), Salvius (Romaine), Cedar (Oakleaf), and Adriana (Butterhead) had the highest yields among each group during summer. Growth chamber study indicated that Dragoon, Adriana, New Fire Red and Red Sails cultivars had higher yields than other cultivars under cooler (10 and 15.5 °C) air temperature conditions. In the winter study, lettuce cultivars did not reach harvestable size even after 40 days of growth without SL and root zone heating. Supplemental light composition significantly affected lettuce growth with higher yield under Purple (with higher proportion of red) than White LED lighting. Commercially acceptable lettuce could be produced using root zone heating. In general, plants grown under CFT yielded higher than those grown under NFT in the winter trial. Among the cultivars, Salvius, Black Seeded Simpson, Cedar, and Red Sails performed better under SL and root zone heating during winter.</p>

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.

The group 2 late embryogenesis abundant (LEA) proteins, also known as the dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. In this work, we study the potential roles that dehydrins may have in stabilizing membranes and actin microfilaments during cold stress. We have cloned and expressed in E. coli two dehydrins from Thellungiella salsuginea, denoted TsDHN-1 (acidic) and TsDHN-2 (basic). These proteins were expressed as SUMO-fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and for structural analysis by CD and Fourier transform infrared (FTIR) spectroscopy. We show using transmission-FTIR spectroscopy that ordered secondary structure is induced and stabilized in these proteins by association with large unilamellar vesicles emulating the lipid compositions of plant plasma and organellar membranes. The increase in secondary structure by membrane association is further facilitated by the presence of Zn2+. Lipid composition and temperature have synergistic effects on the secondary structure. Our single molecule force spectroscopy studies also suggest tertiary folding of both TsDHN-1 and TsDHN-2 induced by association with lipids. From Langmuir-Blodgett monolayer compression studies, and from topographic studies using atomic force microscopy at variable temperature, we conclude that TsDHN-1 stabilizes the membrane at lower temperatures. Finally, we show that the conformations of TsDHN-1 and TsDHN-2 are affected by pH, interactions with cations and membranes, and phosphorylation. Actin assembly by these dehydrins was assessed by sedimentation assays, and viewed by transmission electron and atomic force microscopy. Phosphorylation enabled both dehydrins to polymerize actin filaments, a phenomenon that may occur in the cytosols of plant cells undergoing environmental stress. These results support the hypothesis that dehydrins stabilize plant organellar membranes and/or the cytoskeleton in conditions of stress, and further that phosphorylation may be an important feature of this stabilization. / NSERC

Red alder (Alnus rubra Bong.) is the only tree in British Columbia and the Northwest US to engage in actinorhizal symbiosis to fix atmospheric nitrogen. This study was conducted to explore the plasticity in growth and physiology among 58 17-year-old red alder families in response to variation in climate in two common garden plots, one at Bowser, BC and one at Terrace, BC. Physiological assessments included height and diameter growth, bud flush, water use efficiency as measured by δ13C, cold hardiness as measured by controlled freezing and electrolyte leakage, autumn leaf senescence, and instantaneous and seasonally integrated rates of nitrogen fixation as measured by acetylene reduction and natural abundance δ15N isotope analysis, respectively. Significant differences were identified among families for growth (height and diameter), bud burst stage, leaf senescence, cold hardiness, and bud nitrogen content. No significant differences among families were identified for water use efficiency as measured by δ13C, or for rates of nitrogen fixation as measured by either acetylene reduction or natural abundance δ15N. This study identified possible adaptive differences among red alder genotypes, especially in traits such as bud flush timing, cold hardiness, or nitrogen fixation and their respective contributions to growth. These differences often reflected a tradeoff between growth and the ability to tolerate an extreme environment. Cold hardiness results indicate that red alder families are well adapted to their climate of origin, and may not be able to acclimate sufficiently to a northward assisted migration of genotypes. Nitrogen fixation results demonstrated gaps in our current knowledge of Frankia distribution and impact on the actinorhizal symbiosis in British Columbia. / Graduate