Calcium is a component of signal transduction pathways that allow plants to respond to numerous endogenous and environmental signals during growth and development. Calcium-mediated signaling involves multiple components including: 1) channels, pumps, and exchangers that act in concert to generate a change in cytosolic calcium, 2) calcium-binding proteins that sense the calcium change, and 3) downstream target proteins that modify enzyme activity and gene expression needed for the subsequent response. One method for achieving specificity during calcium signaling is through regulation of the calcium-binding proteins that perceive changes in cytosolic calcium. These proteins can be regulated through differences in expression in response to stimuli, localization within the cell or plant, affinity for calcium, and interaction with downstream target proteins; all of which can result in specific cellular responses. My projects have focused on the Arabidopsis thaliana (Arabidopsis) CALCINEURIN B-LIKE10 (CBL10) calcium-binding protein, and specifically on understanding: 1) how post-transcriptional regulation of the CBL10 gene is used to modulate seedling growth in saline conditions (salinity), and 2) CBL10’s function in the flower during growth in salinity. In addition, 3) I have examined the roles of two putative CBL10-interacting proteins in plant growth and development. CBL10 is alternatively spliced into two transcripts; CBL10 encoding the characterized, full-length protein and CBL10 LONG A (CBL10LA) encoding a putative truncated protein due to a pre-mature termination codon within a retained intron. When seedlings are grown in the absence of salinity, both alternatively spliced transcripts are detected; however, in response to salinity, levels of the CBL10LA transcript are reduced. My data suggest a model in which the relative abundance of the two transcripts regulates the SALT-OVERLY-SENSITIVE (SOS) pathway involved in maintaining cellular sodium ion homeostasis. The presence of CBL10LA in the absence of salinity ensures that the SOS pathway is inactive. The removal of CBL10LA in response to saline conditions results in CBL10 activation of the SOS pathway to prevent sodium ions from accumulating to toxic levels in the cytosol. Successful fertilization during flowering requires the coordinated development of stamens and pistils. Stamens must elongate and anthers dehisce to release pollen onto the stigma while the pistil prepares to receive the pollen and promote growth and targeting of the female gametophyte. When the cbl10 mutant is grown in salinity, flowers are sterile due to decreased stamen elongation, reduced anther dehiscence, and abnormal pistil development. My studies demonstrated that the SOS pathway is not involved in maintaining flower development in salinity and indicate that CBL10 functions in different pathways to regulate vegetative and reproductive development during growth in saline conditions. An in silico search for Arabidopsis proteins that might interact with CBL10 resulted in the identification of two components of the Mediator complex involved in the regulation of transcription in eukaryotes. While additional studies I carried out suggest that interaction with CBL10 is unlikely, I have shown that these proteins are important for plant growth in high levels of chloride and in maintenance of growth in short-day conditions.

The Arabidopsis HSP101 protein belongs to the Hsp100/ClpB family of AAA+ proteins (ATPases Associated with various cellular Activities). This family of proteins, in collaboration with the Hsp70 chaperone system, has the remarkable ability to solubilize protein aggregates and refold proteins back to their native forms. Thus, their chaperone activity is necessary for acquired thermotolerance in organisms as diverse as bacteria and plants. My dissertation project focused on understanding the mechanism of HSP101 action using Arabidopsis thaliana as a model system. The first approach used genetics to screen for suppressors of a specific missense mutant allele of HSP101, hot1-4, in order to find interacting cofactor proteins or key substrates of HSP101, or other processes involved in thermotolerance. Four extragenic suppressors that can overcome the heat-hypersensitive phenotype caused by the hot1-4 mutation were isolated and one of them (shot1) was identified as a mutation in a mitochondrion-targeted protein. Although shot1 mutations do not directly interfere with HSP101 function, they reveal independent mechanisms required for thermotolerance, which involve reduced oxidative stress. The second approach used to investigate HSP101 function was to affinity-purify HSP101 and identify associated proteins. For this purpose, transgenic Arabidopsis plants were generated expressing affinity-tagged wild-type and mutant variants of HSP101. As predicted, cytosolic Hsp70s were identified as an interacting partner of HSP101. Surprisingly, 26S proteasome regulatory subunits were also identified, suggesting a possible link between the protein degradation and reactivation pathways. Further experiments were also undertaken to define the importance of different domains of HSP101, as well as the localization of HSP101. Transgenic Arabidopsis plants expressing N- or C-terminally truncated HSP101 indicate that the N-terminal domain of HSP101 is required for full activity in protecting plants from heat stress. However, in contrast to the yeast ortholog, Hsp104, the C-terminal extension of HSP101 was found to be completely dispensable for thermotolerance of Arabidopsis. Additional transgenic plants expressing an HSP101-GFP were also characterized. Initial microscopic analysis confirms nuclear/cytoplasmic localization as has been reported previously for yeast Hsp104. However, the dynamics of subcellular redistribution upon heat stress need to be further investigated to fully understand the potential significance of the observed localization.

Estudo fitoquímico dos extratos de folhas e galhos de Peperomia villipefiola (\"hierba de la plata\"), coletada no Peru, resultou no isolamento e caracterização da miristicina (3-metoxi-4,5-metilenedioxi-alil-benzene) e sete novos cromenos. Os cromenos tiveram suas estruturas elucidadas através de análises espectroscópicas como sendo: 5-hidroxi-7-metil-2,2-dimetil-2H-1-cromeno-6-carboxilato de metila (PVL1), 7-metil-5-metoxi-2,2-dimetil-2H-1-cromeno-8-carboxilato de metila (PVL2), 7-hydroxy-5-metil-2,2-dimetil-2H-1-cromeno-6-carboxilato de metila (PVL3), 5-metil-7-metoxi-2,2-dimetil-2H-1-cromeno-6-carboxilato de metila (PVL4), ácido 7-hidroxi-5-metanol-2,2-dimetil-2H-1-cromeno-6-carboxílico (PVL5), ácido 5-metanol-7-metoxi-2,2-dimetil-2H-1-cromeno-6-carboxílico (PVL6) e 5-acetoximetanol-7-hidroxi-2,2-dimetil-2H-1-cromeno-6-carboxilato de metila (PVL7). Uma proposta biossintética foi sugerida, no qual o anel aromático seria formado pela via policetídica enquanto que a porção isoprênica poderia ser oriunda tanto da via do mevalonato quanto da via do fosfato de metileritriol. / The leaves and stems extracts from the Peperomia villipetiola (\"herb of the silver\"), collected in Peru, yielded myristicine (3-methoxy-4,5-methy/enedioxy-allylbenzene) and seven new chromenes. The chromenes, all new compounds, had their structures elucidated by means of spectroscopic analysis as: methyl 5-hydroxy-7-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (PVL1), methyl 5-methoxy-7-methyl-2,2-dimethyl-2H-1-chromene-8-carboxylate (PVL2), methyl 7-hydroxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (PVL3), methyl 7-methoxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (PVL4), 5-methanol7-hydroxy-2 ,2-dimethyl-2H-1-chromene-6-carboxylic acid (PVL5), 5-methanol-7-methoxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (PVL6), and methyl 5-acetoxymethanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate (PVL7). A biosynthetic scheme was suggested in which the aromatic ring should be formed by polyketide pathway while the isoprenyl moiety either from mevalonic acid or methylerythriol phosphate pathway.

Maxixe Paulista é um novo tipo de maxixe obtido a partir do cruzamento de Cucumis anguria x Cucumis longipes. Após ciclos de seleção massal intercalados a ciclos de endogamia, foram obtidas linhagens que diferem do tipo comum pelas suas características de ausência de espiculosidade, tamanho de fruto e formato de folha não lobulada semelhante ao pepino. Em três ensaios de campo, linhagens e híbridos de Maxixe Paulista foram avaliados quanto ao comportamento e produção de frutos em três sistemas de cultivo: a) com cobertura de polietileno e fertirrigação; b) cultivados em vasos com substrato, com tutoramento e podas e em ambiente protegido; c) no sistema de cultivo tutorado em rede agrícola. No primeiro ensaio, foram avaliadas dez linhagens de Maxixe Paulista e uma cultivar do Maxixe Comum. As linhagens de Maxixe Paulista apresentaram um peso médio de fruto de 66 a 91% maior que o tipo Comum. A produção em peso total de frutos não diferiu entre os dois tipos de maxixe. O cultivo em canteiros com cobertura de polietileno e fertirrigação por gotejamento proporcionou uma produtividade estimada de 51,89t.ha -1 . No segundo ensaio, três linhagens do Maxixe Paulista e o tipo Comum foram cultivados em vasos com substrato, em ambiente protegido, com tutoramento e podas. As linhagens de Maxixe Paulista apresentaram um peso médio de fruto de 62 a 84% maior que o tipo Comum. A linhagem 2 foi tão produtiva, em termo de peso total, quanto o tipo Comum porém seu peso médio de fruto foi 75% maior. A planta de maxixe mostrou ser inadequada para condução no protocolo de tutoramento e podas da cultura do pepino. No terceiro ensaio, foram avaliadas quatro linhagens e seis híbridos simples, quanto ao comportamento e produção de frutos no sistema de cultivo tutorado em rede agrícola. A produção e a qualidade dos frutos dos híbridos foi equivalente a das linhagens. A rede agrícola se mostrou adequada para o cultivo de Maxixe Paulista na forma tutorada. A concentração da frutificação ocorreu nas hastes secundárias e terciárias, cujas gavinhas mantiveram as plantas presas à rede. Esta técnica de condução facilitou a colheita e incrementou a qualidade dos frutos. / Paulista Gherkin is a new gherkin type derived from Cucumis anguria x Cucumis longipes. After mass selection cycles intercalated with inbreeding cycles, lines were obtained that differ from the common types by their greater fruit size, spine abscence and non-lobular shaped leaves, similar to cucumber ones. In three experiments, Paulista gherkin lines and hybrids were evaluated for their yield and fruit production in three production systems: a) under polyethylene mulching and fertigation crop system; b) trellised and pruned plants grown in pots with substrate under high plastic tunnel; c) under the trellised net crop system. In the first experiment, ten lines of Paulista Gherkin and one type of Common Gherkin were evaluated. Paulista Gherkin fruits were, in average, 66 to 91% heavier than the Common Gherkin ones. Total fruit weight, of two gherkin types did not differ. Polyethylene-covered bed and drip fertigation use promoted an estimate yield of 51,89 t.ha -1 . In the second experiment, three Paulista Gherkin lines and Common Gherkin were cultivated in pots with substrate, under high plastic tunnel and they were trellised and pruned. Paulista Gherkin fruits were, in average, 62 to 84% heavier than the Common Gherkin ones. Paulista Gherkin line number 2 was productive as the Common type, considering the total fruits weight. However, its average fruit weight was 75% higher. Gherkin plants showed to be inadequate for to be conducted as suggested for the greenhouse cucumber crop protocol. In the third experiment, four lines and six single hybrids performance were evaluated for their yield and fruit production under trellised net crop system. Hybrids and lines were similar for yield and fruit quality. The trellised net crop system showed to be suitable to support Paulista Gherkin plants. Fruit concentration occurred in the secondary and tertiary lateral branch and plants was hold by tendrils on the net. Trellised gherkin in net mate harvest easier and enhance fruit quality.

É conhecido que a utilização repetitiva e consecutiva dos mesmos herbicidas, ou de herbicidas diferentes, porém com mesmo mecanismo de ação, exerce uma pressão de seleção sobre as populações de plantas daninhas controladas. As duas formas principais de resposta das populações de plantas daninhas resultantes da pressão de seleção dos herbicidas são a mudança da flora pela seleção de espécies de plantas daninhas tolerantes, ou a seleção de biótipos de plantas daninhas resistentes aos herbicidas. Na prática têm sido observadas, pelos produtores de cana-de-açúcar, mudanças de flora pela seleção de algumas espécies de capim-colchão (Digitaria spp), as quais não têm sido controladas eficientemente pelos herbicidas normalmente recomendados para o seu controle. Suspeita-se que estas populações selecionadas são constituídas por diferentes espécies de capim-colchão, que apresentam níveis maiores de tolerância aos herbicidas. Sendo assim, os objetivos desta pesquisa foram: (i) identificar taxonomicamente as espécies de capim-colchão infestante da cultura da cana-de-açúcar no Estado de São Paulo, e avaliar a eficácia de herbicidas no controle de Digitaria nuda a nível de campo; (ii) comparar a suscetibilidade das espécies D. nuda e D. ciliaris aos herbicidas do grupo químico das triazinas, uréias substituídas, imidazolinonas, triazinonas e aos herbicidas, cujo mecanismo de ação é a inibição da síntese de caroteno, e quantificar o nível de tolerância das espécies através da elaboração de curvas de dose-resposta; (iii) determinar, com a utilização de 14C-herbicida, a absorção e translocação pelas espécies Digitaria nuda e Digitaria ciliaris dos herbicidas diuron (via folha) e imazapyr e metribuzin (via raiz). A identificação das espécies de capim-colchão (Digitaria spp) foi efetuada seguindo chave de identificação existente na literatura. No estudo da eficácia de herbicidas no controle de Digitaria nuda a nível de campo, foram utilizados oito tratamentos herbicidas, aplicados em condições de pré-emergência. As avaliações de controle foram realizadas aos 15, 30 e 60 dias após a aplicação (DAA). O estudo da eficácia de herbicidas no controle das espécies Digitaria nuda e Digitaria ciliaris, foram conduzidos em casa-de-vegetação. Foram utilizados oito tratamentos herbicidas, aplicados em condições de pré-emergência. As avaliações de controle foram realizadas aos 14 e 21 (DAA). O estudo do nível de tolerância foi realizado em casa-de-vegetação do Departamento de Produção Vegetal da ESALQ/USP, Piracicaba/SP, através de curvas de dose-resposta. Os estudos de absorção e translocação foram conduzidos em câmara de crescimento, e as análises realizadas no Laboratório de Ecotoxicologia do Centro de Energia Nuclear na Agricultura (CENA/USP), em Piracicaba, SP, utilizando-se 14C-herbicida, medindo-se a radioatividade em diferentes partes das plantas. Os resultados permitiram concluir que: (i) a espécie Digitaria nuda foi selecionada pela aplicação repetitiva dos herbicidas utilizados no controle de capim-colchão na cultura da cana-de-açúcar; demonstrando ser mais tolerante aos herbicidas dos grupos químicos das imidazolinonas e uréias substituídas, quando comparada à Digitaria ciliaris; (ii) a partir das curvas de dose-resposta, pode ser verificado que a Digitaria nuda demonstrou ser mais tolerante que a Digitaria ciliaris aos herbicidas diuron, imazapyr e tebuthiuron; (iii) a absorção e translocação não foram os mecanismos responsáveis pela tolerância apresentada pela Digitaria nuda aos herbicidas diuron e imazapyr. / It is known that the repetitive and successive use of the same or differents herbicides, but with same mechanism of action, impose a selection pressure on the weed population controled. The two main forms of weed population response resulted from the selection pressure of the herbicide is, weed species shifts, through selection of the tolerant species of weed, or selection of resistant biotipes within the population. It has been observed by sugarcane growers weed species shifts whitin the genus (Digitaria spp). It is suspected that the populations selected are constituted by different crabgrass species, with higher level of herbicide tolerance. The objectives of the research were: (i) identify taxonomically the crabgrass infesting the sugarcane crop in São Paulo state – Brazil, and to study herbicide efficacy in the control of the Digitaria nuda in field conditions; (ii) compare the susceptibility of D. nuda and D. ciliaris to herbicides of the chemical groups triazine, substituted urea, imidazolinone, triazinone, and inhibitors of carotene synthesis, and quantify the level of susceptibility of the species, through dose-response curves; (iii) determine, by using 14C-herbicide, the absorption and translocation by the species Digitaria nuda and Digitaria ciliaris of herbicides diuron (by leaf), and imazapyr and metribuzin (by root). The identification of the crabgrass species (Digitaria spp) was carry out following a identification key published in the literature. In the study of herbicides efficacy in the control of Digitaria nuda in field conditions, eight herbicide treatments, applied in pre-emergence were used, and the control percentage was evaluated at 15, 30 and 60 days after herbicide application (DAA). The study of herbicide efficacy to control the species Digitaria nuda and Digitaria ciliaris were conducted in greenhouse. Eight herbicide treatments, applied in pre-emergence were used, and the control percentage was evaluated at 14 and 21 days after herbicide application (DAA). The study of the level of tolerance was conducted in the greenhouse of the Department of Crop Science, University of São Paulo, Piracicaba/SP – Brazil, through dose-response curves; The study of absorption and translocation were conducted in a growth chamber, and the 14C-herbicide analysis was done in the Laboratory of Ecotoxicology of the Center of Nuclear Energy Applied to Agriculture, Piracicaba/SP, Brazil, using 14C-herbicide, measuring radioactivity in different plant parts. The results allowed to conclud that: (i) the specie Digitaria nuda was selected by repetitive applications of the herbicides used in the control of the crabgrass in the sugarcane crop, showing that the specie is more tolerant to the herbicides of the chemical group of imidazolinone and substituted urea, when compared to Digitaria ciliaris; (ii) From the dose-response curves study, it was observed that Digitaria nuda showed more tolerance than Digitaria ciliaris to the herbicides diuron, imazapyr and tebuthiuron; (iii) The absorption and translocation were not the mechanism responsible for the tolerance showed by Digitaria nuda to the herbicides diuron and imazapyr.

Maxixe Paulista é um novo tipo de maxixe obtido a partir do cruzamento de Cucumis anguria x Cucumis longipes. Após ciclos de seleção massal intercalados a ciclos de endogamia, foram obtidas linhagens que diferem do tipo comum pelas suas características de ausência de espiculosidade, tamanho de fruto e formato de folha não lobulada semelhante ao pepino. Em três ensaios de campo, linhagens e híbridos de Maxixe Paulista foram avaliados quanto ao comportamento e produção de frutos em três sistemas de cultivo: a) com cobertura de polietileno e fertirrigação; b) cultivados em vasos com substrato, com tutoramento e podas e em ambiente protegido; c) no sistema de cultivo tutorado em rede agrícola. No primeiro ensaio, foram avaliadas dez linhagens de Maxixe Paulista e uma cultivar do Maxixe Comum. As linhagens de Maxixe Paulista apresentaram um peso médio de fruto de 66 a 91% maior que o tipo Comum. A produção em peso total de frutos não diferiu entre os dois tipos de maxixe. O cultivo em canteiros com cobertura de polietileno e fertirrigação por gotejamento proporcionou uma produtividade estimada de 51,89t.ha -1 . No segundo ensaio, três linhagens do Maxixe Paulista e o tipo Comum foram cultivados em vasos com substrato, em ambiente protegido, com tutoramento e podas. As linhagens de Maxixe Paulista apresentaram um peso médio de fruto de 62 a 84% maior que o tipo Comum. A linhagem 2 foi tão produtiva, em termo de peso total, quanto o tipo Comum porém seu peso médio de fruto foi 75% maior. A planta de maxixe mostrou ser inadequada para condução no protocolo de tutoramento e podas da cultura do pepino. No terceiro ensaio, foram avaliadas quatro linhagens e seis híbridos simples, quanto ao comportamento e produção de frutos no sistema de cultivo tutorado em rede agrícola. A produção e a qualidade dos frutos dos híbridos foi equivalente a das linhagens. A rede agrícola se mostrou adequada para o cultivo de Maxixe Paulista na forma tutorada. A concentração da frutificação ocorreu nas hastes secundárias e terciárias, cujas gavinhas mantiveram as plantas presas à rede. Esta técnica de condução facilitou a colheita e incrementou a qualidade dos frutos. / Paulista Gherkin is a new gherkin type derived from Cucumis anguria x Cucumis longipes. After mass selection cycles intercalated with inbreeding cycles, lines were obtained that differ from the common types by their greater fruit size, spine abscence and non-lobular shaped leaves, similar to cucumber ones. In three experiments, Paulista gherkin lines and hybrids were evaluated for their yield and fruit production in three production systems: a) under polyethylene mulching and fertigation crop system; b) trellised and pruned plants grown in pots with substrate under high plastic tunnel; c) under the trellised net crop system. In the first experiment, ten lines of Paulista Gherkin and one type of Common Gherkin were evaluated. Paulista Gherkin fruits were, in average, 66 to 91% heavier than the Common Gherkin ones. Total fruit weight, of two gherkin types did not differ. Polyethylene-covered bed and drip fertigation use promoted an estimate yield of 51,89 t.ha -1 . In the second experiment, three Paulista Gherkin lines and Common Gherkin were cultivated in pots with substrate, under high plastic tunnel and they were trellised and pruned. Paulista Gherkin fruits were, in average, 62 to 84% heavier than the Common Gherkin ones. Paulista Gherkin line number 2 was productive as the Common type, considering the total fruits weight. However, its average fruit weight was 75% higher. Gherkin plants showed to be inadequate for to be conducted as suggested for the greenhouse cucumber crop protocol. In the third experiment, four lines and six single hybrids performance were evaluated for their yield and fruit production under trellised net crop system. Hybrids and lines were similar for yield and fruit quality. The trellised net crop system showed to be suitable to support Paulista Gherkin plants. Fruit concentration occurred in the secondary and tertiary lateral branch and plants was hold by tendrils on the net. Trellised gherkin in net mate harvest easier and enhance fruit quality.

Dry bean (<i>Phaseolus vulgaris</i>) is currently being introduced for rainfed production in the Dark Brown soil zone in Saskatchewan. Insufficient water is one of the major factors limiting crop production in this soil zone. Ten pinto bean genotypes (Othello, Agate, GH196-2, 6315, 5325, Earliray, Fiesta, ISB82-354, UI 111 and Nodak) were exposed to drought stress in the field to study their rooting pattern under drought stress. Three pinto bean genotypes (Othello, Agate and GH196-2) with an extensive root system in the deeper soil layers and three (Earliray, Fiesta and ISB82-354) with a less extensive root system were exposed to drought stress and nonstress conditions in the growth room and in the field to study the relationship between rooting pattern and seed yield. In addition, three indeterminate pinto bean genotypes (Othello, Nodak and Fiesta) and three determinate genotypes (Agate, Agassiz and Earliray) were exposed to a soil moisture gradient to study the effects of soil moisture level on seed yield and other agronomic traits. Othello, Agate and GH196-2 had an extensive root system in the deeper soil layers, whereas Earliray, Fiesta and ISB82-354 had a less extensive root system in the deeper soil layers. An extensive root system in the deeper soil layers was associated with increased soil moisture depletion at those depths. Shoot dry mass, total root dry mass, mean root length density and root:shoot ratio were the same for the ten pinto bean genotypes. Pinto bean genotypes with an extensive root system (Othello, Agate and GH196-2) in the deeper soil layers had higher seed yield compared to genotypes with a less extensive root system (Earliray, Fiesta and ISB82-354) in the deeper soil layers in the field and in the growth room. Due to insufficient drought stress in the field, the rooting pattern of the six pinto bean genotypes was the same under irrigation and rainfed conditions. In the growth room, pinto bean genotypes with an extensive root system showed a stronger recuperative ability after a severe drought stress compared to genotypes with a less extensive root system. Root traits were positively correlated with seed yield under adequate soil moisture or where drought stress was relieved before maturity. Soil moisture deficit reduced number of pods per plant, plant height, seed weight, seed yield and seed nitrogen concentration of both determinate and indeterminate pinto bean genotypes. Indeterminate pinto bean genotypes had, on average, higher mean seed yield and showed a greater seed yield response to increasing soil moisture availability compared to determinate genotypes. However, the difference in seed yield of the two groups was small under high drought stress, except in the case of one very early maturing determinate genotype.

Recent research has focused on the molecular characterization of null waxy (Wx), 'Wx-A1b, Wx-B1b', and 'Wx-D1b', alleles that produce no detectable Wx proteins in the endosperm starch of allohexaploid wheat (<i>Triticum aestivum</i> L.; 2n = 6x = 42; AABBDD). The major objectives of this thesis were to (1) isolate and characterize a Wx wheat cDNA and (2) to identify aberrant 'Wx' transcripts encoded by the null 'Wx-A1b' allele of CDC Wx2, a waxy hexaploid wheat line, which result in an absent Wx-A1 protein (~59 kD). In the first study, a cDNA library prepared from developing wheat kernels (cv. Fielder) was screened using a homologous PCR-digoxigenin labeled wheat cDNA probe. A 2.2 kb cDNA clone denoted GBSSIMMI (Accession no. Y16340) was sequenced and identified as encoding a Wx-D1 protein. The deduced amino acid sequence showed 94% similarity with a wheat Wx-A1 peptide, 96% similarity with a wheat Wx-B1 peptide, and 100% identity with two wheat Wx-D1 peptides. A 33-nucleotide deletion, encoding 11 amino acids (AMLCRAVPRRA), was detected within the GBSSIMMI cDNA relative to a previously isolated wheat cDNA (accession no. X57233). Complementation analysis using a glycogen synthase deficient 'E. coli' strain and an 'in vitro' starch synthase assay did not indicate that GBSSIMMI encoded a functional Wx-D1 protein. In the second study, two sister lines CDC Wx2 and CDC Wx6 were obtained by crossing lines Bai-Huo (carries null 'Wx-D1b' allele; lacks Wx-D1 protein) and Kanto 107 (carries null 'Wx-A1b' and -'B1b' alleles; lacks Wx-A1 and -B1 proteins). Waxy protein profiling, amylose concentration determinations, Northern blot analysis, and reverse transcriptase PCR (RT-PCR) analysis were conducted. Ten RT-PCR derived cDNA clones were selected from each genotype and characterized by DNA sequencing analyses. The waxy phenotype of CDC Wx2, lacking Wx-A1, -B1, and -D1 proteins and possessing a reduced amylose concentration ~4%), was associated with dramatically reduced levels of a 2.4 kb 'Wx' transcript when compared to the higher levels in a wildtype control line. DNA sequencing of clones from Kanto 107 and CDC Wx2 characterized two types of aberrant 'Wx' transcripts, one containing intron 1 and another containing introns 1 and 4. Intron 1 in both types of aberrant 'Wx' transcripts contained a premature stop codon which resulted in the translation of a truncated Wx protein ~4 or 11 kD). Analysis of CDC Wx6, lacking Wx-B1 and -D1 proteins and possessing a reduced amylose concentration (~14%) failed to reveal aberrant ' Wx' transcripts, suggesting that the RNA defects in this study were not responsible for the absence of the Wx-B1 or -D1 proteins. Thus, the aberrant Wx transcripts were encoded by the null 'Wx-A1b' allele. The presence of a premature stop codon in the 'Wx' transcripts encoded by the null 'Wx-A1b' allele explained the absence of the ~59 kD Wx-A1 protein in CDC Wx2 and its parental line Kanto 107.

The spatial structure of white spruce populations was studied in 52 stands. White spruce tree density increased with age in the 200-year chronosequence after fire. Tree height and DBH peaked at about 120 years after fire. Sapling density along the chronosequence after clearcutting exhibited similar pattern to that after fire, but peaked earlier. White spruce seedlings were present in various densities and heights along the chronosequence after fire, producing uneven-aged stands. Seedling regeneration was mostly on the LFH layer (72%) in younger plots and on logs (97%) in old plots. Seedlings in both regeneration types were evenly spaced at a young age. This pattern changed to random and clumped in older stands. Artificially planted clearcuts formed more even-aged stands. Physiological, morphological and growth responses to sun and shade treatments in the greenhouse were examined in white spruce seedlings collected from three naturally-regenerated (N1, N2 and N3) and three field-planted (P1, P2, and P3) stands. Dark respiration and light compensation points declined by 70 and 81% respectively, in shade- compared to sun-acclimated seedlings. Quantum yield, total chlorophyll content, specific leaf area and absolute water content increased by 45, 33, 32 and 50% respectively, in response to shade treatment. Height was not affected by light regime. Fewer and longer secondary branches were noticed in the shade compared to full sun. At light saturation, populations P1 and N3 showed similar photosynthetic responses under both light regimes (around 6 [mu]mol m-2 s-1). Populations P2, P3 and N2 performed more poorly in the sun than in the shade (8.2, 8.7 and 9.1 in shade, versus 5.1, 4.1 and 5.5 in full sun, respectively). Photosynthetic rate in N1 was greater in full sun than in shade (14.7 and 11.1 [mu]mol m -2 s-1, respectively). Differences in physiological responses to light among populations suggest the presence of more than one ecotype. The variation in physiological and morphological parameters within field-planted and naturally-regenerated populations was large, and did not show any obvious differences among populations. RAPD analysis showed abundant polymorphism in all populations. The naturally-regenerated arid the field-planted populations demonstrated similar within and among regeneration-type variation. Of the total genetic variation 82.9% was due to intra-population variation, while inter-population variation and regeneration type accounted for 16.7 and 0.4% of the total variation, respectively. It appears that selection pressure during reforestation was not great enough to cause a significant decline in the genetic diversity of field-planted compared to naturally-regenerated white spruce.

Genetic variation exists in <i>Raphanus</i> that could be of use to <i>Brassica</i> breeders. Of particular interest is the Ogura system of cytoplasmic male sterility (CMS) which has been worked on extensively in a <i>Brassica napus</i> background. Problems have been experienced in <i>B. napus</i>restorer lines due to the inheritance of a large segment of <i>Raphanus</i> chromosome containing the fertility restoring locus. This restorer introgression is located on the <i>Brassica</i> C genome making it only of use for <i>B. napus</i> and not for <i>B. rapa</i> or <i>B. juncea</i>. This thesis describes the development of the materials necessary for the introgression into the <i>Brassica</i> A genome of a defined segment of <i>Raphanus</i> chromosome containing a restorer locus. Defined genetic stocks of <i>Raphanus</i> were developed that contained specific loci controlling restoration of Ogura CMS. This material was used to develop populations segregating for specific restorer loci. Extensive RFLP maps of three <i>Raphanus</i> populations were developed and aligned, resulting in a robust consensus map of the entire <i>Raphanus</i> genome. Three restorer loci were accurately mapped on three separate linkage groups. The segment of <i>Raphanus</i> that is implicated in the restoration of Ogura CMS in a <i>B. napus</i> restorer line developed by INRA was identified and it did not correspond to any of the regions containing the three mapped restorer loci, suggesting the presence of more restorer loci in <i>Raphanus</i>. Comparative mapping between the <i>Raphanus</i> genome map and previously generated <i>Brassica</i> A genome RFLP maps demonstrated large regions of collinearity between segments of chromosomes of the two species. Preliminary examination of the two genome maps suggest they contain essentially the same overall genetic content but with large segments of the genomes rearranged with respect to each other. Likely sites of <i>Raphanus</i> restorer introgression into the <i>Brassica</i> A genome were predicted. Trigenomic tetraploids were developed in which pairing and recombination between homoeologous segments of <i>Raphanus</i> and <i>Brassica</i> A chromosomes should result. Progeny of these individuals will allow an assessment of the pattern and extent of recombination that occurs between the chromosomes of the <i>Raphanus</i> and <i>Brassica</i> A genomes and should lead to the development of 'B. napus' lines carrying Ogura CMS restorer alleles from <i>Raphanus</i>.