Reinhard Jetter

The cuticles covering aerial organs of most plants contain a complex mixture of fatty acid-derived waxes, with various chain lengths and diverse functional groups. These waxes can form crystals which then dictate the physical properties of the surface, by accumulating either ubiquitous compounds or specialty components that are restricted to certain taxa. Relatively little is known about the biosynthesis of the specialty compounds, and whether unrelated species utilize the same mechanisms to form them. However, specialty wax biosynthesis must be elucidated to understand the formation of wax crystals and, therefore, the mechanisms underlying plant protection against drought and other stresses.The goal of my thesis is to further our understanding of the biosynthesis of specialty compounds ketones and β-diketones. Specifically, I aimed to: (i) carry out in-depth wax analyses of co-occurring compound classes, homologs and isomers with implications for possible biosynthetic mechanisms; (ii) identify and characterize enzymes involved in β-diketone formation based on comparative analysis of β-diketones and related compounds in closely and remotely related plant species. This work was done on species that were confirmed or suspected to comprise ketones and β-diketones. Furthermore, the species were chosen for their importance as food crops (Allium fistulosum, Triticum aestivum), ornamental plants (Dianthus species) or biofuels (Panicum virgatum).To achieve the first goal, I analyzed and compared the wax compositions of Allium fistulosum wild type and a wax-deficient mutant, which allowed for elucidation of the mechanism for ketone and ketol formation by carbon chain elongation, in contrast to the previously described hydroxylation mechanism. For the waxes of Dianthus species, I found characteristic chain length profiles that had implications for the mechanisms underlying β-diketone production. To achieve the second goal, I carried out wax analyses of several species across Poaceae subfamilies to investigate the ubiquity of their biosynthetic mechanisms. This was followed by characterization and functionalization of the main enzymes involved in β-diketone production.Overall, this work highlights the importance of understating the underlying mechanisms behind mid-chain functional compound production and the effects those mechanisms have on chain-length, deposition and therefore crystal formation.

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The plant cuticle plays important roles in organ development and adaptation to stresses. Cuticular waxes are mixtures of very-long-chain fatty acid derivatives, also comprising cyclic compounds. The biosynthesis of the common wax constituents and specialty compounds occurring in some plant species has been researched. However, little is known about how mid-chain hydroxyls may also be introduced during hydrocarbon chain formation by Claisen condensations. In my Ph.D studies, I first focused on the wax composition and biosynthesis of specialty wax compounds of Drimys winteri. In Chapter 3, I first investigate the morphology and composition of the Drimys winteri leaf surface. The stomatal plugs on the abaxial leaf surface consist of cutin polymer with sub-micron cavities and tubule-shaped wax crystals above the cutin. I also found the major Drimys winteri wax compound, nonacosan-10-ol, likely has its functional group established by Claisen condensation before the hydrocarbon chain is fully formed, and not by hydroxylation afterwards. The wax mixture also comprised various alkanediols, alkanetriols and ketols with characteristic constellations of hydroxyl groups, suggesting P450 enzymes act alone and in tandem to hydroxylate both secondary alcohols and their diol derivatives. In Chapter 4, methyl ketones, 2-alkanols and δ-lactones were identified as minor constituents of the wax mixture on the Drimys winteri leaves, and their chain length distributions imply characteristic chain length patterns of intermediates derived from the fatty acid elongation complexes involved. In Chapter 5, I focus on the crucial condensing enzymes which catalyze Claisen condensations growing the hydrocarbon chains into very-long-chain fatty acyls. For this, I used the model plant Arabidopsis to investigate the roles of condensing enzymes β-ketoacyl-CoA synthases (KCSs). Mutants lacking one of these enzymes, KCS3, displayed sepal fusions, chain length shifts in the cuticular waxes of several organs, and altered guard cell surface structures. A similar enzyme KCS12, had redundant effects on sepal fusion and bud wax composition with KCS3. In contrast, mutation of the KCS19 led to seed fusions and altered chain length in suberin of seed coats. Overall, all three KCSs were thus found involved in forming very-long-chain compounds coating various Arabidopsis organ surfaces.

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Plant cuticles play important roles in plant development, fertility, and adaptation. The cuticular waxes directly facing the environment are made mainly from derivatives of very-long-chain (VLC) aliphatics. Previous studies on cuticular waxes clarified the biosynthesis of ubiquitous wax components based on the model plant Arabidopsis thaliana. However, the biosynthesis and chemical structures of numerous uncommon wax components from different plant species are still unclear. In my Ph.D. studies, I focused on wax components from both Arabidopsis thaliana and Poaceae crop species to identify new enzymes involved in plant cuticular wax biosynthesis and further characterize their biochemical functions. In chapter 2, I investigated alkane biosynthesis in bread wheat. The linkage of alkane content and transcriptome data revealed a candidate gene, TaCER1-1A. The phenotype of corresponding nullisomic-tetrasomic substitution wheat lines and heterologous expression of TaCER1-1A in rice and Arabidopsis confirmed its function in alkane synthesis. In chapter 3, the products associated with β-diketones in barley were profiled. New diketone homologs, the 2-alkanol ester profile and the ¹³C isotope abundance of different wax components indicated the unique biosynthesis pathway to β-diketones. The biochemical functions of the core enzymes on this pathway, DIKETONE METABOLISM HYDROLASE (DMH) and DIKETONE METABOLISM POLYKETIDE SYNTHASE (DMP), were investigated. In vivo and in vitro assays showed the direct condensation between 3-ketoacid and fatty acyl-CoA catalyzed by DMP, thus revealing the unique character of DMP as a PKS family member and revised the model of the β-diketone synthesis pathway in barley.In chapter 4, VLC alkenes from Arabidopsis young leaves were analyzed. Different from other plant species, alkenes ranging from C₃₃ to C₃₉ with predominance of 7- and 9- isomers were found in Arabidopsis. An acyl-CoA desaturase family member, ADS4.2, was shown to influence alkene formation. ADS4.2 was strongly expressed in young rosette leaves, especially in trichomes, and yeast expression revealed that this enzyme had ω-7 regio-specificity with high preference for acyl-CoAs longer than C₃₂. Wax-synthesis-deficient mutants were used to put the alkene-forming pathway in context with the wax synthesis. The results show that Arabidopsis produces characteristic alkenes through a unique elongation-desaturation pathway with the participation of ADS4.2.

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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.

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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.

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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.

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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.

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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.

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