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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2021)
Plant cuticles seal above-ground organs against non-stomatal water loss, and therefore are vital for survival on land. Besides providing a transpiration barrier, cuticles have important secondary functions, for example to protect from harmful UV radiation, and to provide a self-cleaning mechanism and mechanical support. Cuticles consist of aliphatic very-long-chain wax compounds (C₂₄ to C₃₈) and a cutin polymer. The diversity of cuticular wax compositions across the plant kingdom but also between different organs and ontogenetic stages is remarkable, yet the regulating mechanisms and function of those chemical differences are largely unknown. In the study presented here, a new approach was used, increasing temporal and spatial resolution and integration of chemical wax analyses with analyses of gene expression patterns of wax biosynthesis genes, using Arabidopsis thaliana as a model organism. The aims of the study were to, first, monitor wax composition and gene expression as a function of leaf development as well as different epidermal cell types and, second, to use this information to identify new wax biosynthesis genes.In the second chapter, high temporal resolution was used to follow the dynamics of wax chemistry and gene expression during development of Arabidopsis thaliana leaves, and I was able to link changes in wax chemistry to differential expression of the elongation enzyme KCS6/CER6.In the third chapter, wax analyses and gene expression data with high spatial resolution were acquired, and I identified differences between Arabidopsis epidermal cell types in wax composition and gene expression. Trichomes had a higher abundance of longer chain waxes (C₃₂ to C₃₈) compared to pavement cells, and the KCS5, KCS8 and KCS16 elongation enzymes were identified as candidates for the elongation of C₃₄₊ waxes. In the fourth chapter, I characterized the Arabidopsis condensing enzyme KCS16 and was able to show that it is functioning on the wax elongation pathway, elongating C₃₄ to C₃₈ acyl-CoA wax precursors, mainly in trichomes but also in pavement cells.
The cuticle is an external barrier of aerial plant organs that prevents desiccation. It is composed of hydrophobic waxes, i.e. complex mixtures of very-long-chain aliphatics, alicyclics and aromatics, lying on top of (epicuticular) and in between (intracuticular) a polyester matrix known as cutin. Wax compositions vary greatly between plant species, organs and tissues, both qualitatively and quantitatively. This thesis describes the identification and quantification of cuticular waxes of three plant species, including structure elucidation of novel compounds, chain length profiling, and wax compound distributions between intracuticular and epicuticular compartments for the first two species.Leaves of Aloe arborescens were found covered with 15 μg/cm² wax on the adaxial side and 36 μg/cm² on the abaxial side, with 3:2 and 1:1 ratios between epicuticular and intracuticular wax layers on each side, respectively. Along with ubiquitous wax compounds, three homologous series were identified as novel 3-hydroxy fatty acids (predominantly C₂₈), their methyl esters (predominantly C₂₈), and 2-alkanols (predominantly C₃₁), and their biosynthetic pathways were hypothesized based on structural similarities and homolog distributions.The adaxial side of young and old Phyllostachys aurea leaves was found covered with 1.7 to 1.9 μg/cm² each of epi- and intracuticular waxes. In addition to typical aliphatics and alicyclics, novel primary amides were identified, with their chain length profile peaking at C₃₀, and found exclusively in the epicuticular waxes, hence near the true plant surface.Flag leaves and peduncles of Triticum aestivum cv. Bethlehem were found covered with 16 and 49 μg/cm² wax, respectively, dominated by 1-alkanols in the case of the former and β-diketones and hydroxy-β-diketones for the latter. Along with previously reported wax classes, numerous new classes were identified as homologous series: 2-alkanol esters, benzyl esters, phenethyl esters, p-hydroxyphenethyl esters, secondary alcohols, primary/secondary diols and their esters, hydroxy- and oxo-2-alkanol esters, 4-alkylbutan-4-olides, internally methyl-branched alkanes, and 2,4-ketols. Other new compounds were found as single homologs: C₃₃ 2,4-diketone, C₃₁ mid-chain β-ketols, C₃₀ mid-chain α-ketols and α-diketone, as well as C₃₁ mid-chain ketones. Biosynthetic pathways are proposed in the thesis for the new compounds, based on common structural features and matching chain length patterns between related compound classes.
Plants coat themselves in a cuticle to hinder transpiration across the vast surface areas they require for photosynthesis. The cuticle is made of cutin, a polyester, and cuticular waxes, aliphatic compounds that form the water barrier.Cuticles of model plants had been major targets for cuticular wax research, and knowledge of their surfaces is relatively advanced. However, this knowledge still does not answer crucial questions about relationships between wax structure and function. Limited studies of non-model species had provided glimpses of a much greater wax chemical structural diversity than that present on model plants. These also had hinted that diverse wax coverages and compositions exist on the surfaces of different species and different plant organs. Before relationships between structure and function can be established, the major dimensions of wax diversity must be described in more detail.To contribute, I aimed to describe wax structural and biological diversity in some model and non-model plant species. I examined the structural diversity of wax compound aliphatic tails and determined that branched compounds on Arabidopsis leaves are iso-branched and that certain wax biosynthesis enzymes can exhibit bias towards or against branched substrates. I also studied functional group diversity by performing a comprehensive literature search to codify our knowledge of wax compounds with secondary functions.I furthered our knowledge of wax coverage and composition on diverse biological surfaces by determining the structures of novel wax compounds from the moss Funaria hygrometrica and profiling the waxes from multiple F. hygrometrica surfaces to reveal that these moss cuticles have some similarities to those of flowering plants. By studying developing Arabidopsis leaves I found that their wax composition, but not coverage, is dynamic with time, pointing to functional optimization and synchronous cell expansion and wax production.This work highlights the importance of chain length specificity in wax biosynthesis, though the mechanisms by which such is achieved are unclear. It also confirms that secondary functional groups on wax molecules are installed by a variety of processes, that these are connected with biosynthetic chain length specificity and that both likely influence the physical and water barrier properties of the cuticle.
Plant cuticles form an external barrier, primarily blocking water loss into the desiccating atmosphere but also inhibiting UV and pathogen penetration. Cuticles consist of hydrophobic wax – a complex mixture of very-long-chain aliphatics and alicyclics – on top of (epicuticular) and in between (intracuticular) the biopolymer cutin. Absolute and relative wax composition varies between species and organs. Considering the diversity of compounds and barrier functions, the question arises: How does each wax compound shape each function? This dissertation presents advancements using Cosmos bipinnatus and Arabidopsis thaliana in three research areas ranging from localization of compounds at the organ, cellular, and sub-cellular levels, to structural elucidation of novel compounds, and finally to functional characterization.Waxes from C. bipinnatus petals, stems, and leaves were shown to be distinct. Petal wax comprised mainly primary alcohols as well as novel 1,2- and 1,3-diols and ketols. These classes were dominated by C₂₂ and C ₂₄ chain lengths. The water resistances of the adaxial (3.0±0.3 x 10⁴ s/m) and abaxial (1.5±0.2 x 10⁴ s/m) surfaces of these petals were lower than average literature values for leaves but similar to fruit, suggesting that the wax composition on ephemeral organs creates a compromised water barrier. Lateral wax heterogeneity was shown for trichomes, which tended to have longer compound chain lengths and a higher percentage of alkanes, as compared to pavement cells in both leaves and stems of A. thaliana. Moreover, a meta-analysis synthesizing the epicuticular and intracuticular wax compositions of all species investigated to date showed vertical wax heterogeneity. Noticeably, cyclic compounds preferentially accumulated in intracuticular wax. This finding was confirmed by over-expression of AtLUP4 in Arabidopsis, which caused β-amyrin accumulation in the intracuticular but not epicuticular wax layer. The presence of β-amyrin reduced the intracuticular wax-caused water resistance (2.4±0.2 x 10³ s/m) to three-quarters of the control (3.4±0.5 x 10³ s/m) while the epicuticular wax resistance for the over-expressor (6.8±0.6 x 10³ s/m) equaled that of the control (6.6±0.9 x 10³ s/m).An understanding of how wax constituents affect cuticular functions will aid in breeding and designing plants capable of withstanding adverse biotic and abiotic conditions.
The biosynthesis of wax components containing secondary functional groups was investigated in the current study. Two fundamentally different pathways were proposed to introduce the secondary functional groups. One pathway involves hydroxylation of elongated substrates. Wax components characterized by two functional groups located on or near the centre of the carbon chain, nonacosane-14,15-diol, -14,16-diol and -13,15-diol as well as corresponding ketols were identified for the first time in Arabidopsis stem wax. The alkanediols and ketols were dominated by the C-14,15 isomers. The absence of alkanediols and ketols in Arabidopsis mah1 mutants that are deficient in secondary alcohol biosynthesis confirmed the biosynthetic relationship between secondary alcohols and alkanediols/ketols (Chapter 3). In pea (Pisum sativum) leaf wax, two novel compound classes were identified as primary/secondary alcohols dominated by octacosane-1,14-diol and secondary/secondary alkanediols hentriacontane-9,16-diol, -8,15-diol and -10,17-diol. Co-localization of the secondary/secondary alkanediols and hentriacontan-15-ol and -16-ol pointed to a biosynthetic relationship (Chapter 4). The diverse structures of compounds identified in the current study suggested that hydroxylases can use substrates other than alkanes. The predominance of isomers within homologues indicated a regiospecificity of the hydroxylases involved in wax biosynthesis. In addition to hydroxylation, secondary functional groups could also be introduced through elongation of carbon chains. Homologous series of 5-hydroxyaldehydes (C₂₄ and C₂₆-C₃₆) and 1,5-alkanediols (C₂₈-C₃₈) were identified in yew (Taxus baccata) needle wax. The relative position of both functional groups suggested that these two compound classes are biosynthetically related and their secondary functional groups are introduced during elongation (Chapter 5). The results of incubation of ¹⁴C-labeled malonyl-CoA and acyl-CoAs with different chain lengths in the presence of California poppy (Eschscholzia californica) microsomes provided the first evidence to support the elongation hypothesis. The results indicated that a carbonyl group rather than a hydroxyl group is introduced during elongation. To provide molecular tools for further investigations of the hypothetical pathway, three full length cDNAs encoding putative KCSs were cloned and one of them, PKCSI, was functionally characterized.
Master's Student Supervision (2010 - 2020)
The epicuticular waxes covering the uppermost organs of the barley plant, including spikes, peduncles and flag leaf sheaths, are dominated by β-diketones and related hydroxy-β-diketones. Cultivars characterized by higher amounts of these compounds display improved resistance to water loss, therefore resulting in higher production yields. It was recently shown that biosynthesis of the different β-diketones and hydroxy-β-diketones relies on (1) a hydrolase known to participate in the formation of β-keto fatty acids, (2) a type-III polyketide synthase involved in β-diketone formation, and (3) a cytochrome P450 believed to perform the final hydroxylation of β-diketones. So far, only the hydrolase has been partially characterized, and the mechanism through which these enzymes form the different β-diketone-related compounds remains to be demonstrated. Here, a detailed chemical analysis of the β-diketone-related compounds found in the wax of barley cv. Morex spikes has revealed the presence of a novel β-diketone whose structure cannot be explained with the currently assumed biosynthesis pathways. In addition, a natural isotope abundance analysis of the predominant barley β-diketone revealed ¹³C enrichment also conflicting with previous biosynthesis hypotheses and prompting detailed studies of the polyketide synthase involved in β-diketone formation. This barley diketone metabolism polyketide synthase, DMP, was characterized in yeast and in vitro, revealing that it catalyzes the formation of β-diketones via head-to-head condensation between β-ketoacids and fatty acyl-CoAs. In the light of further results confirming that the first pathway enzyme, diketone metabolism hydrolase (DMH), delivers mainly C₁₆ β-ketoacid as substrate for DMP, a revised pathway for β-diketone biosynthesis in barley may now be proposed. Overall, the contribution of this research to the knowledge of barley β-diketones biosynthesis represents an important step towards the breeding of new cultivars with enhanced drought resistance and higher yield production. Interestingly, it seems very likely that current findings for β-diketone biosynthesis can be extrapolated from barley to wheat as a very closely related species, ultimately also enabling the breeding of stress-resistant wheat cultivars.
5-Alkylresorcinols are a class of phenolic lipids which have been identified in the cuticular waxes of various cereal crops. Due to their antifungal and antibacterial properties, ARs have potential applications as nutraceuticals. They are biosynthesized by type-III polyketide synthases (PKSs). Two candidate PKS genes were previously isolated from the two model grass species Secale cereale and Brachypodium distachyon, and were shown to encode alkylresorcinol synthases (ScARS and BdARS, respectively). Here I report the further characterization of these two enzymes, with the goal to test whether they are involved in the formation of cuticular wax alkylresorcinols. Series of alkylresorcinols were identified and quantified, containing ARs with C₁₉-C₂₇ alkyl chains in S. cereale waxes, and C₁₇-C₂₅ on B. distachyon waxes. In addition, a new series of methyl-branched alkylresorcinols was identified with C₁₉-C₂₅ chains. The accumulation of ARs was monitored in waxes on various organs of etiolated and normal plants, and the product amounts found to correlate with the expression patterns of the putative ARS genes in each species. Subcellular localization using GFP fusions showed that the ARS proteins are associated with ER membranes of epidermal cells, where very-long-chain acyl CoA substrates of ARSs are known to accumulate. Overall, my data indicate that both enzymes are indeed involved in the biosynthesis of grass surface alkylresorcinols.
The plant cuticle is a hydrophobic layer that seals the surface of primary aerial organs of terrestrial plants and serves in protecting the tissues from abiotic and biotic stresses. Lipids are synthesized in the plastid and in the endoplasmic reticulum (ER) of epidermal cells for eventual export and deposition on the surface. Great progress has been made by genetic studies in the model plant Arabidopsis thaliana in elucidating fatty acid elongation, but knowledge of alkane biosynthesis is still scarce. The current work was focused on expanding our current understanding of alkane biosynthesis in Arabidopsis thaliana. A recent discovery of Susceptible to Coronatine-Deficient Pst DC3118-2 (SCD2) whose mutant has a leaf-specific increase in aldehydes and a decrease in alkanes suggests that SCD2 has a role in converting aldehydes to alkanes. In this thesis, further characterization of SCD2 revealed that alkanes are decreased in two mutant lines, the wax of mutants was restored by transgene complementation with the native gene, the transcript is abundant in leaves, and the promoter is active in the phloem of vasculature. Finally, the protein localized to the ER, consistent with its role in wax biosynthesis. This work provided evidence for yet another gene whose product is involved in formation of cuticular alkanes in Arabidopsis thaliana.Double mutants were generated to further study wax biosynthesis in both stems and leaves. The cer1cer3 mutant had greatly reduced total stem and leaf wax amounts compared to wild-type, as well as a substantial reduction of alkanes. It has an increase in C30 primary alcohol levels like the cer3 parent, indicating epistasis. This suggests that CER3 precedes CER1 in alkane formation. Furthermore, it is severely male-sterile with a reduction in epicuticular wax crystals. Wax biosynthesis is similar in stems and leaves of cer1cer3, cer1cer4 and cer3cer4. The cer1cer3 will be an important tool to test domain functionality of CER1 and CER3 and may shed more light on the mechanisms of alkane formation in Arabidopsis thaliana.
Plant cuticles are the interface for plant-environment interactions, and the first barrier protecting plants from environmental stresses such as water loss and pathogens. Structurally, the cuticle consists of a hydrophobic polymer lattice, cutin, and cuticular waxes deposited inside and outside cutin. The major components of the cuticular waxes are aliphatics derived from very-long-chain (VLC) fatty acids, such as alkanes and aldehydes. Besides compounds with primary functionalities, some compounds with two or more functional groups have also been identified in cuticular waxes. However, only limited knowledge about them has been acquired so far. The current work was to identify novel 1,2- and 1,3-bifunctional wax compounds from various plant species, in order to expand our current understanding of their structure, biosynthesis and function in plant cuticles. Synthetic methods were first developed to produce various VLC 1,2- and 1,3-bifunctionalized standard compounds for current and future structure elucidation studies. Unknown compounds found in Cosmos bipinnatus petal wax were identified as alkane-1,2-diol monoacetates by GC-MS, with chain lengths ranging from C20 to C24. The ratio between the primary and the secondary monoacetates was quantified to be 3:5, as opposed to the thermodynamic equilibrium ratio of 7:3. Novel β-hydroxy acid methyl esters were also identified from Aloe arborescens leaf wax, with chain lengths ranging from C26 to C30. In addition, two NMR-based methods were established to study the stereoconfigurations of alkane-1,2-diols from C. bipinnatus petal wax, and the carbons bearing secondary hydroxyl functionality were determined to have predominately the R-configuration.Apart from the research on bifunctional compounds, synthetic methods to produce β-deuterium labeled VLC substrates were also established. The resulting C30 fatty acid methyl ester was double-labeled and can be used directly or indirectly as substrates in future biochemical assays. At last, the hypothetic substrate for the CER1 enzyme implicated in wax alkane biosynthesis, C30 aldehyde, was synthesized and used in in vivo assays with heterologously expressed protein.
Alkylresorcinols are phenolic lipids which occur in diverse plant species as well as microorganisms. In plants, alkylresorcinols are usually deposited at or near the surfaces where they are thought to serve as a first line of defense. Earlier work in our lab had shown the surface accumulation of alkylresorcinols in Secale cereale leaves was mainly restricted to the cuticle. However, direct evidence showing the protective role of these bioactive compounds at the surface is still insufficient. The current work was to investigate the biosynthesis of cuticular alkylresorcinols in order to get a better understanding of their biological function. This research focused on S. cereale, since it had previously been shown to contain relatively large amounts of alkylresorcinols, and on Brachypodium distachyon, a closely related genetic model system with completely sequenced genome. First, chemical analyses revealed that the cuticular wax covering leaves of B. distachyon included 5% of alkylresorcinols with alkyl chains varying from C₁₇ to C₂₅. Therefore, it was hypothesized that both species have genes encoding alkylresorcinol synthases (ARSs). A central goal of this work was to clone and characterize potential ARSs. One ARS (BdARS) was cloned from B. distachyon by mining the Brachypodium expressed sequence tag libraries and one ARS (ScARS) was cloned from S. cereale using a homology-based cloning strategy. In vivo biochemical characterization in yeast Saccharomyces cerevisiae demonstrated that both enzymes were capable of using C₁₀ to C₂₂ fatty acyl-CoAs with malonyl-CoA to generate a broad range of alkylresorcinols. Organ-specific expression in leaves but not in roots was observed for both BdARS and ScARS. Additionally, the expression pattern of ScARS matched the time-course of cuticular alkylresorcinol accumulation along the leaf of S. cereale. An investigation into their subcellular localization revealed that both ARSs were likely localized to the endoplasmic reticulum membrane. All these results taken together support the idea that BdARS and ScARS are the enzymes responsible for the biosynthesis of cuticular alkylresorcinols, and that the cuticular alkylresorcinols are indeed biosynthesized for a protective function associated with the wax lining the surface of grass leaves.
Alkylresorcinols (ARs) are bioactive compounds found in 11 plant families. Indirect evidence showed that they were likely near/at the surface of plant organs, suggesting their defensive role against biotic and abiotic stressors in the environment. However, neither the exact function nor the forming mechanisms are known. To assess whether their primary function is exerted at plant surface and to unravel their biosynthesis, the local distribution of ARs has to be determined first. Hence, the goals of the research were to analyze the ARs in the leaf waxes, to compare their amounts with interior concentrations, to determine AR distribution within the surface wax layers, and to monitor their accumulation as a function of leaf development. Rye (Secale cereale L.) was chosen for this investigation, since previous studies had indicated that it has high levels of ARs in various organs. The total wax mixture firstly extracted consisted of primary alcohols (71%), alkyl esters (11%), aldehydes (5%), and small amounts of alkanes, steroids, secondary alcohols, fatty acids and unknown compounds. ARs were identified by GC-MS and comparison with nonadecylresorcinol (AR19:0). They contributed 3% of total wax, and comprised a homologous series with odd-numbered alkyl side chains from C19 to C27. Secondly, abaxial and adaxial waxes separately sampled, contained very similar relative quantities of all constituents. Thirdly, the epicuticular and intracuticular wax layers were selectively extracted. ARs comprised 2% of the intracuticular wax, yet none in the epicuticular wax. All other wax components were spread uniformly between both wax layers. By analyzing various segments at four growth stages of rye leaves, the spatial distribution of waxes along the length of leaf blade and wax accumulation over time were assessed. All the major compound classes shared similar wax production zone, spatial distribution pattern and timing for wax production. Yet ARs were formed in a remarkably different spatial area and time periods. They were only detectable at later growth stages (IV and III) and not detected near POE in contrast to major wax constituents which were produced from each growth stage to its next adjacent stage and mainly within 2-cm segments beyond POE.