No description available.

We wanted to develop rapid and cost-effective drought tolerance screening methods for mass amounts of germplasm. In 2009 and 2010, we evaluated sixty-two maize inbred lines and their hybrid testcross progeny using seedling stress response and epicuticular wax accumulation as predictors of drought tolerance. The seedling screening method measured germination, survival and recovery percentages after a series of drought cycles in a greenhouse environment. Eight inbred lines had significantly (P < 0.05) lower germination than the mean estimate, but hybrid testcrosses were not significantly different. The second-to-last day of survival cycle and the second day of recovery cycle best explained genotypic differences for inbred lines and hybrid testcrosses respectively. One inbred line performed well as both an inbred line and hybrid testcross, but poor correlation over the sample set (R2 = 0.0097) was observed. Flag leaves taken at flowering from plants under full and limited irrigation regimes were sampled for epicuticular wax. Extracted wax weight for genotypes was compared as a percentage of leaf weight (percent wxlfwt) and leaf area (percent wxwta). Eleven genotypes had above average percent wxlfwt as both inbred lines and hybrid testcrosses. Thirteen genotypes had above average percent wxwta as either inbred lines or hybrid testcrosses. Irrigation treatment was not significant (P > 0.05). Heritability of percent wxlfwt was 0.17 (inbred lines) and 0.58 (hybrid testcrosses). Heritability of percent wxwta was 0.41 (inbred lines) and 0.59 (hybrid testcrosses). Correlations (R2) for percent wxlfwt and percent wxwta were 0.19 and 0.03, respectively. Heritability of grams of grain per ear and total grain yield was highest in hybrid testcrosses, with no correlation between inbreds and hybrids. The developed seedling screening method easily allowed visible drought tolerance observations in inbred lines and hybrid testcrosses but does not seem heritable in our germplasm. Epicuticular wax weight is not an ideal primary trait to evaluate for drought tolerance, but may be a good candidate to observe as a secondary trait in relation to grain yield production in hybrids. Results from this research best supports breeder selection of hybrid germplasm using seedling drought tolerance in conjunction with epicuticular wax.

Crops adapted to dry conditions are essential to meet future food, feed and energy needs. Knowledge of interaction between drought tolerance traits and their response to varying water supply conditions would improve selection for yield stability traits. This study focused on determining the association between the QTL regulating the staygreen trait in sorghum with improved canopy temperature depression (CTD) as regulated by total and compositional epicuticular wax content in a recombinant inbred line population derived from BTx642 and RTx7000. Phenotypic data were collected in 3 replicated field trials and 1 greenhouse trial. Plants with higher leaf EWL had cooler canopies. Our results also confirmed that staygreen genotypes are able to maintain cooler canopy than the non-stay-green genotypes under drought and hot conditions. We have suggested that wax might offer a more stable indicator for selection of drought tolerance under a variety of weather conditions. Composite interval mapping identified a total of 28 QTL, fifteen of which had significant overlaps. The overlap between QTL for cuticular leaf wax and QTL for staygreen exhibits a departure from the QTL overlaps for other traits with that of cuticular leaf wax. We have also suggested that under drought stress, the QTL for staygreen may be expressed earlier in time (at anthesis) than had been previously believed.

Epicuticular waxes form a layer on the outer surface of all land plants and play a fundamental role in their interaction with the environment. Development of a rapid and accurate method for the characterization of these waxes could allow the use of wax composition as a novel phenotype during plant breeding and selection. In this study, the suitability of Direct Analysis in Real Time Mass Spectrometry (DART-MS) for the characterization of epicuticular waxes was investigated. This method provides a “fingerprint” of the relative abundance of all constituents in the analyte based on mass, is suitable for very high throughput, requires minimal sample preparation, and is able to provide for the characterization of even complex biological mixtures. Herein, the suitability of DART-MS for analysis of epicuticular wax was investigated through analysis of samples of extracted wax collected from the flag leaves of a 279 line association mapping population grown across four environments with two replicates in both irrigated and drought treatments. Additionally, for a subset of samples, wax was collected from glumes in order to test for differences in wax composition between tissue types. In all, a total of 3,454 wax extracts were analyzed with three technical replicates. The above analysis generated a total of 13,164 mass spectra (“fingerprints”), made at an average rate of 30 seconds each. Multivariate analyses including random forest, principal component analysis, and linear discriminant analysis, were used to identify the presence of differences between the spectra of wax from different tissues (leaves/glumes) and treatment types (irrigated/drought). The peaks best serving as predictors of sample class for each comparison were examined, and tentative identifications were made through comparison of the associated mass with literature and publicly available databases. As a separate test of concept, the ability to distinguish between the epicuticular wax compositions of individual varieties was determined through analysis of a group of closely related lines developed at CIMMYT which differed in agronomic performance.

Master of Science / Department of Biology / Loretta Johnson / Plant epicuticular wax (ECW) isolates internal tissues from harsh external conditions increasing drought tolerance. Beta-diketone-rich ECW reflect light and result in a glaucous phenotype that may ameliorate the thermal environment of the leaf. The overall goal is to characterize the form and function of ECW in two closely related, but phenotypically divergent grasses. [italicized]Andropogon gerardii ssp. [italicized]gerardii, big bluestem, is a non-glaucous, agronomically and ecologically dominant grass in the United States while [italicized]Andropogon gerardii ssp. [italicized]hallii, sand bluestem, is a glaucous subspecies restricted to dry, sandy soils. The objectives are to contrast sand and big bluestem ECW chemistry, morphology, and physiology to determine the distinctions in ECW resulting in the glaucous phenotype and determine the effect this has on leaf optical qualities and permeability. Gas chromatography mass spectrometry (GC-MS) and scanning electron microscopy (SEM) were used to examine ECW chemistry and micromorphology. It was hypothesized that beta-diketones and beta-diketone tubules where present only in leaves of sand bluestem and that the ECW was more reflective and abundant and the cuticle was less permeable. Beta-diketones and tubular ECW were absent in big bluestem and common on sand bluestem’s surface, although less than 20% of ECW was beta-diketones. Functional implications of ECW phenotypes were investigated by comparing minimum conductance (G[subscript]min), wax load, reflectance, and transmittance. Reflectance, with and without ECW, and G [subscript]min were measured with an infrared gas analyzer and a spectroradiometer, respectively. Sand bluestem had twice the ECW in mg cm[superscript]2 (P=.01) and three times lower G [subscript]min in ms[superscript]-1 10[superscript]-5 (P=.02). Partial least squares (PLS) models were trained to predict subspecies from reflectance spectra and were able to distinguish the subspecies. These experiments indicate that in comparison to big bluestem, increased reflectance is a property uniquely imparted to sand bluestem by ECW and the presence of beta-diketones determines the distinction. Glaucous crop species have shown higher yield under drought and extreme weather, including drought, is expected to become more common. Therefore, this study of glaucous waxes, may be applied in engineering drought tolerance.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers.

Analysis of cuticular wax from Kalanchoe daigremontiana leaves was performed to identify the constituent components within the wax, determine how these changed during leaf ontogenesis, and discover how they were distributed within the cuticle. Analysis of extracted cuticular wax by gas chromatography, mass spectrometry, and comparison with authentic standards led to the identification of triterpenoids including glutinol, friedelin, germanicol, epifriedelanol, glutinol acetate and β-amyrin as well as very long chain fatty acid (VLCFA) derivatives including alkanes, primary alcohols, aldehydes, fatty acids, and alkyl esters. Cuticular wax composition in young K. daigremontiana leaves was dominated by triterpenoids, which made up over 70% of the lipid soluble compounds. During leaf ontogenesis, wax composition changed to include a higher proportion of VLCFA derivatives, which made up approximately 50% of cuticular wax in mature leaves. The most abundant triterpenoids in the wax were glutinol and friedelin, both fairly uncommon pentacyclic triterpenoids with a complex proposed biosynthetic mechanism. Tritriacontane (C33 alkane) was the most abundant compound within the VLCFA derivatives. Cuticular wax accumulation was found to correspond well to leaf growth, with both processes slowing at the same time. Variations in the ratio of friedelin-like compounds to glutinol-like compounds during leaf ontogenesis suggest the presence more than one active triterpenoid synthase enzyme in the leaves of K. daigremontiana. VLCFA compounds were found mainly in the epicuticular wax on both the adaxial and abaxial surfaces, while triterpenoids were relatively more abundant in the intracuticular layer. Two different epicuticular wax crystal forms were observed by scanning electron microscopy (SEM) which can be described as platelets with sinuate margins and twisted ribbons. Based on SEM and chemical data as well as previous reports of crystal composition, it is hypothesized that each crystal type has a unique composition, with the platelets containing one or more triterpenoids and the twisted ribbons containing alkanes and other VLCFA derivatives. Confirmation of this hypothesis will have to await further investigation. This research provides information that will aid in the larger goals of characterizing a glutinol or friedelin synthase and understanding the gradients established within epicuticular and intracuticular wax layers. / Science, Faculty of / Chemistry, Department of / Graduate

<p> Previous work on the ultrastructure of leaf surfaces has been confined to commercial terrestrial plants. Until recently there was a conflicting overlap of definitions of surface structures. Lately, studies have concentrated on the role of the epicuticular wax layer in surface phenomena such as water permeability, transpiration, and herbicide susceptibility. The initiating factors in surface wax formation and the mode of extrusion still remain unresolved. </p> <p> An emergent hydrophyte, Zizania aquatica L., was selected to attempt to clarify the initiation and extrusion of epicuticular wax. The first appearance of epicuticular wax occurs while the whole plant is submerged. The wax platelet shape is probably controlled by an endogenous circadian rhythm with very little environmental control. The significance of water depth, temperature, continous light, physical and chemical abrasion are discussed in terms of the surface morphology of the three types of leaves. </p> / Thesis / Master of Science (MSc)

A soja é a cultura agrícola com maior extensão de área plantada no Brasil, país que se destaca no cenário mundial como o segundo maior produtor e exportador desta oleaginosa. A ferrugem asiática (FA), causada pelo fungo Phakopsora pachyrhizi, apresenta-se como um dos mais graves problemas fitossanitários da cultura da soja no Brasil, principalmente por não existirem, até o presente momento, cultivares com níveis de resistência satisfatórios. Com o objetivo de melhor se conhecer a biologia de Phakopsora pachyrhizi e alguns fatores relacionados ao processo infeccioso em soja, no presente trabalho foram avaliados: (1) Métodos de preservação de urediniósporos; (2) Influência da descontinuidade do molhamento foliar no processo infeccioso dos urediniósporos; (3) Influência da luminosidade e da superfície foliar no processo infeccioso de urediniósporos de P. pachyrhizi; (4) Influência do estádio fenológico da soja na infecção de P. pachyrhizi. Os resultados obtidos mostraram que a desidratação dos esporos proporcionou maior viabilidade destes ao longo do tempo, para todas as condições de armazenamento testadas. No deep freezer foi possível preservar os esporos por até 231 dias, independente da sua condição de hidratação. No ambiente, os esporos não desidratados e desidratados permaneceram viáveis por 16 e 22 dias, respectivamente. A reversão de dormência dos esporos foi efetiva ao empregar hidratação (24h de câmara úmida) seguida ou não por choque térmico (40&#176;C/5 minutos). Em todos os tratamentos onde se aplicou molhamento foliar descontínuo, a severidade de FA foi sempre inferior quando comparada ao molhamento contínuo, principalmente quando a interrupção do molhamento se deu após 4 horas de câmara úmida inicial. A interrupção temporária do molhamento afetou os esporos que haviam germinado, pois os mesmo não foram aptos a infectar após novo período de molhamento. Com relação à luminosidade, os urediniósporos foram aptos a infectar plantas de soja tanto na luz quanto no escuro. Porém, severidade maior foi observada quando se inoculou a superfície adaxial, com posterior incubação das plantas no escuro. Os experimentos in vitro mostraram que, na ausência de luz, houve maior germinação e maior formação de apressórios. O teor de cera epicuticular e o seu aspecto ultra-estrutural não apresentaram diferenças entre as superfícies adaxial e abaxial no cultivar BRS 154. Os cultivares BRS 154 e BRS 258, no estádio fenológico reprodutivo, R5, apresentaram menor severidade em relação aos estádios V3 e R1. O período latente médio (PLM) da FA foi 8 e 9 dias para os cultivares BRS 154 e BRS 258, respectivamente. O PLM não se diferenciou em função do estádio fenológico para ambos cultivares. Quanto à influência da idade da folha na suscetibilidade à FA, a folha 1, considerando-se o sentido base-ápice da planta, mostrou maior suscetibilidade e freqüência de infecção de P. pachyrhizi . Os resultados apresentados são essenciais, tanto por disponibilizarem informações sobre a biologia e epidemiologia do fungo, quanto por servirem como base para novos estudos, nas mais diversas áreas sobre este importante patossistema. / Soybean is the agricultural crop with the largest planted area in Brazil. The country stands out in the world scenario as the second-largest producer and exporter of this oilseed crop. The Asian rust (AR), caused by the fungus Phakopsora pachyrhizi, is one of the most serious phytosanitary problems of soybean in Brazil, especially because no cultivars exist so far with satisfactory resistance levels. In order to acquire better knowledge about the biology of Phakopsora pachyrhizi and some factors related to its infectious process in soybean, the following were evaluated in the present work: (1) Uredospore preservation methods; (2) Influence of leaf wetness discontinuity on the infectious process of uredospores; (3) Influence of luminosity and leaf surface on the infectious process of P. pachyrhizi uredospores; (4) Influence of phenological stage of soybean on infection by P. pachyrhizi . The results obtained showed that spore dehydration provided greater spore viability with time, in all storage conditions tested. Spores could be preserved for up to 231 days in the deep freezer, regardless of their hydration condition. At room temperature, non-dehydrated and dehydrated spores remained viable for 16 and 22 days, respectively. Spore dormancy reversion was effective when hydration was used (24h in humid chamber), followed or not by thermal shock (40&#176;C/5 minutes). In all treatments where discontinuous leaf wetting was applied, AR severity was always lower when compared with continuous wetting, especially when wetting was interrupted after 4 hours of initial treatment in the humid chamber. Temporary wetness interruption affected spores that had already germinated, as these were not able to infect after a new wetness period. As to luminosity, uredospores were capable of infecting soybean plants both in the light and in the dark. However, higher severity was observed when the adaxial surface was inoculated, with later incubation of the plants in the dark. The in vitro experiments showed that there was greater germination and greater formation of appressoria in the absence of light. Epicuticular wax content and its ultrastructural aspect did not show differences between the adaxial and abaxial surfaces in cultivar BRS 154. At the R5 reproductive phenological stage, cultivars BRS 154 and BRS 258 showed smaller severity in relation to stages V3 and R1. The AR mean latent period (MLP) was 8 and 9 days for cultivars BRS 154 and BRS 258, respectively. MLP differences due to phenological stage were not detected in any of the cultivars. As to the influence of leaf age on AR susceptibility, leaf 1 showed greater susceptibility and frequency of infection by P. pachyrhizi, considering the plant base-apex direction. The results herein presented are essential, either because they make information available on the biology and epidemiology of the fungus, or because they serve as a foundation for new studies on the most diverse areas about this important pathosystem.