Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present.

Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present.

Cuticular wax is a component of the plant cuticle, the lipid barrier which covers the surface of above ground primary plant organs and plays important protective roles. The isolation of wax deficient mutants from Arabidopsis and other plants resulted in identification and isolation of genes required for wax deposition, and broadened our knowledge of this process in plants. To identify additional components involved in cuticular wax production, I investigated the role of the XTC1 gene, defective in the xtcl (extra cotyledon 1) mutant. This mutant was reported to have reduced levels of cuticular wax on its inflorescence stems and accumulate a large number of oil bodies in the primordia of its extra cotyledons. Stem wax extraction and gas chromatography analysis showed that the total xtcl stem wax is decreased 3-fold in comparison to the wild type, and that all wax components were reduced to a similar extent. Compositional analyses of leaf and seed fatty acids demonstrated that saturated fatty acid content was decreased by around 55%, and unsaturated fatty acid content was approximately 20% lower in xtcl mutants. A detailed examination of xtcl seeds revealed seed deformities, altered seed coat permeability and defective seed mucilage extrusion. Positional cloning of the XTC1 gene resulted in the discovery that it is identical to FATB, an already characterized gene known to encode the fatty acid thioesterase B. The FATB enzyme releases saturated free fatty acids (C16:0and C18:0) from ACP in the plastid and allows their export across the plastid envelope. Analysis of FATB gene expression pattern showed that FATB is transcribed ubiquitously in all tissues and in different development stages. It is therefore not surprising that FATB disruption results in multiple lipid associated phenotypes, including decreased cuticular wax amounts and altered fatty acid compositions of leaves and seeds. Additional phenotypes caused by mutations in FATB that affect embryo and seed development and lead to appearance of extra cotyledons, altered permeability of the seed coat and defective seed mucilage extrusion are difficult to explain at present. / Science, Faculty of / Botany, Department of / Graduate

In wheat, cooler canopies have been associated with yield under high temperature stress. The objectives of this study were, i) to understand the role of leaf cuticular waxes as physiological adaptive mechanisms during reproductive stage high temperature stress ii) define quantitative trait loci (QTL) regulating leaf cuticular waxes and determine its link with the QTL for reproductive stage heat tolerance iii) define stable QTL associated with leaf cuticular waxes and yield stability across environments. For the first objective, thirteen wheat cultivars were subjected to a 2-day heat treatment at 38 degrees C at 10 days after pollination (DAP). Leaf cuticular waxes, canopy temperature depression and stomatal conductance were estimated during high temperature stress. At maturity the percent reduction in yield components in each cultivar was calculated. The wheat cultivars 'Kauz' and 'Halberd' had significantly high leaf cuticular wax content of 2.91mg/dm^-2 and 2.36mg/dm^-2 respectively and cooler canopies. Leaf cuticular waxes were significantly correlated with leaf temperature depression and reduction in yield components. A set of 121 recombinant inbred lines (RIL) population derived from the cross of heat tolerant wheat cultivar 'Halberd' and heat susceptible wheat cultivar 'Karl 92' was utilized for QTL mapping. The RIL population received a 2-day short-term high temperature stress at 38°C at 10DAP in 2008 and a long-term high temperature stress at 38 degrees C from 10DAP until maturity in 2009 in the greenhouse. The RIL population was also planted in College Station, Texas in 2009 and 2010 and in Uvalde, Texas in 2010. Leaf cuticular wax was estimated at 10DAP and leaf/spike temperatures were recorded during grain filling. Yield components were estimated after harvest. Heat susceptibility indexes for main spike yield components were estimated in the greenhouse. Overall ten significant QTL were identified for leaf cuticular waxes each explaining 8-19 percent of the variation respectively. Stable QTL for leaf cuticular waxes were located on chromosome 5A and 1B and co-localized with QTL for leaf/spike temperature depression and HSI for kernel weight and single kernel weight of main spike. Another QTL on chromosome 1B contributed by Karl92 was found in the greenhouse and field environments and co-localized with a previously identified QTL on 1B for spike non-glaucousness. The results suggest that leaf cuticular waxes may reduce leaf temperatures and improve adaptation during high temperature stress.

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

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