Dynamic changes to the plant cuticle include the production of volatile cuticular wax-derived compounds.

The cuticle is a hydrophobic structure that seals plant aerial surfaces from the surrounding environment. To better understand how cuticular wax composition changes over development, we conducted an untargeted screen of leaf surface lipids from black cottonwood (Populus trichocarpa). We observed major shifts to the lipid profile across development, from a phenolic and terpene-dominated profile in young leaves to an aliphatic wax-dominated profile in mature leaves. Contrary to the general pattern, levels of aliphatic cis-9-alkenes decreased in older leaves following their accumulation. A thorough examination revealed that the decrease in cis-9-alkenes was accompanied by a concomitant increase in aldehydes, one of them being the volatile compound nonanal. By applying exogenous alkenes to P. trichocarpa leaves, we show that unsaturated waxes in the cuticle undergo spontaneous oxidative cleavage to generate aldehydes and that this process occurs similarly in other alkene-accumulating systems such as balsam poplar (Populus balsamifera) leaves and corn (Zea mays) silk. Moreover, we show that the production of cuticular wax-derived compounds can be extended to other wax components. In bread wheat (Triticum aestivum), 9-hydroxy-14,16-hentriacontanedione likely decomposes to generate 2-heptadecanone and 7-octyloxepan-2-one (a caprolactone). These findings highlight an unusual route to the production of plant volatiles that are structurally encoded within cuticular wax precursors. These processes could play a role in modulating ecological interactions and open the possibility for engineering bioactive volatile compounds into plant waxes.

Evolution and molecular basis of substrate specificity in a 3-ketoacyl-CoA synthase gene cluster from Populus trichocarpa.

Very long chain fatty acids (VLCFAs) are precursors to sphingolipids, glycerophospholipids, and plant cuticular waxes. In plants, members of a large 3-ketoacyl-CoA synthase (KCS) gene family catalyze the substrate-specific elongation of VLCFAs. Although it is well understood that KCSs have evolved to use diverse substrates, the underlying molecular determinants of their specificity are still unclear. In this study, we exploited the sequence similarity of a KCS gene cluster from Populus trichocarpa to examine the evolution and molecular determinants of KCS substrate specificity. Functional characterization of five members (PtKCS1, 2, 4, 8, 9) in yeast showed divergent product profiles based on VLCFA length, saturation, and position of the double bond. In addition, homology models, rationally designed chimeras, and site-directed mutants were used to identify two key regions (helix-4 and position 277) as being major determinants of substrate specificity. These results were corroborated with chimeras involving a more distantly related KCS, PtCER6 (the poplar ortholog of the Arabidopsis CER6), and used to show that helix-4 is necessary for the modulatory effect of PtCER2-like5 on KCS substrate specificity. The role of position 277 in limiting product length was further tested by substitution with smaller amino acids, which shifted specificity toward longer products. Finally, treatment with KCS inhibitors (K3 herbicides) showed varying inhibitor sensitivities between the duplicated paralogs despite their sequence similarity. Together, this work sheds light on the molecular mechanisms driving substrate diversification in the KCS family and lays the groundwork for tailoring the production of specific VLCFAs.

Understanding the Role of Populus ECERIFERUM2-Likes in the Biosynthesis of Very-Long-Chain Fatty Acids for Cuticular Waxes.

Cuticular waxes are derived from very-long-chain fatty acid (VLCFA) precursors made by the concerted action of four enzymes that form the fatty acid (FA) elongation complex. The condensing enzyme of the complex confers specificity to substrates of different chain lengths, yet on its own cannot account for the biosynthesis of VLCFAs longer than 28 carbons (C28). Recent evidence from Arabidopsis thaliana points to a synergistic role of clade II BAHD acyltransferases and condensing enzymes in the elongation of VLCFAs beyond C28. In Populus trichocarpa, clade II is composed of seven uncharacterized paralogous genes (PtCER2-like1-7). In the present study, five of these genes were heterologously expressed in yeast and their respective FA profiles were determined. PtCER2-likes differentially altered the accumulation of C28 and C30 FAs when expressed in the presence of the condensing enzyme AtCER6. Among these, PtCER2-like5 produced the highest levels of C28 FAs in yeast and its expression was localized to the epidermis in ß-glucuronidase-reporter poplar lines, consistent with a role in cuticular wax biosynthesis. Complementation of the A. thaliana cer2-5 mutant with PtCER2-like5 increased the levels of C28-derived cuticular waxes at the expense of C30-derived components. Together, these results demonstrate that the role of CER2-likes in cuticular wax biosynthesis is conserved in Populus clade II BAHD acyltransferases.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

Arabidopsis sucrose synthase localization indicates a primary role in sucrose translocation in phloem.

Sucrose synthase (SuSy) is one of two enzyme families capable of catalyzing the first degradative step in sucrose utilization. Several earlier studies examining SuSy mutants in Arabidopsis failed to identify obvious phenotypic abnormalities compared with wild-type plants in normal growth environments, and as such a functional role for SuSy in the previously proposed cellulose biosynthetic process remains unclear. Our study systematically evaluated the precise subcellular localization of all six isoforms of Arabidopsis SuSy via live-cell imaging. We showed that yellow fluorescent protein (YFP)-labeled SuSy1 and SuSy4 were expressed exclusively in phloem companion cells, and the sus1/sus4 double mutant accumulated sucrose under hypoxic conditions. SuSy5 and SuSy6 were found to be parietally localized in sieve elements and restricted only to the cytoplasm. SuSy2 was present in the endosperm and embryo of developing seeds, and SuSy3 was localized to the embryo and leaf stomata. No single isoform of SuSy was detected in developing xylem tissue of elongating stem, the primary site of cellulose deposition in plants. SuSy1 and SuSy4 were also undetectable in the protoxylem tracheary elements, which were induced by the vascular-related transcription factor VND7 during secondary cell wall formation. These findings implicate SuSy in the biological events related to sucrose translocation in phloem.

Exploiting Natural Variation to Uncover an Alkene Biosynthetic Enzyme in Poplar.

Alkenes are linear hydrocarbons with one or more double bonds. Despite their potential as biofuels and precursors for specialty chemicals, the underlying biochemistry and genetics of alkene biosynthesis in plants remain elusive. Here, we report on a screen of natural accessions of poplar (Populus trichocarpa), revealing that the leaf cuticular waxes are predominantly composed of alkanes and alkenes. Interestingly, the accumulation of alkenes increases with leaf development, is limited to the abaxial side of the leaf, and is impaired in a few accessions. Among other genes, a ß-ketoacyl CoA synthase gene (PotriKCS1) was downregulated in leaves from non-alkene-producing accessions. We demonstrated biochemically that PotriKCS1 elongates monounsaturated fatty acids and is responsible for the recruitment of unsaturated substrates to the cuticular wax. Moreover, we found significant associations between the presence of alkenes and tree growth and resistance to leaf spot. These findings highlight the crucial role of cuticular waxes as the first point of contact with the environment, and they provide a foundation for engineering long-chain monounsaturated oils in other species.

A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins.

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.

Evolution of a complex locus for terpene biosynthesis in solanum.

Functional gene clusters, containing two or more genes encoding different enzymes for the same pathway, are sometimes observed in plant genomes, most often when the genes specify the synthesis of specialized defensive metabolites. Here, we show that a cluster of genes in tomato (Solanum lycopersicum; Solanaceae) contains genes for terpene synthases (TPSs) that specify the synthesis of monoterpenes and diterpenes from cis-prenyl diphosphates, substrates that are synthesized by enzymes encoded by cis-prenyl transferase (CPT) genes also located within the same cluster. The monoterpene synthase genes in the cluster likely evolved from a diterpene synthase gene in the cluster by duplication and divergence. In the orthologous cluster in Solanum habrochaites, a new sesquiterpene synthase gene was created by a duplication event of a monoterpene synthase followed by a localized gene conversion event directed by a diterpene synthase gene. The TPS genes in the Solanum cluster encoding cis-prenyl diphosphate-utilizing enzymes are closely related to a tobacco (Nicotiana tabacum; Solanaceae) diterpene synthase encoding Z-abienol synthase (Nt-ABS). Nt-ABS uses the substrate copal-8-ol diphosphate, which is made from the all-trans geranylgeranyl diphosphate by copal-8-ol diphosphate synthase (Nt-CPS2). The Solanum gene cluster also contains an ortholog of Nt-CPS2, but it appears to encode a nonfunctional protein. Thus, the Solanum functional gene cluster evolved by duplication and divergence of TPS genes, together with alterations in substrate specificity to utilize cis-prenyl diphosphates and through the acquisition of CPT genes.

Determination of residues responsible for substrate and product specificity of Solanum habrochaites short-chain cis-prenyltransferases.

Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species.

Adaptive evolution of threonine deaminase in plant defense against insect herbivores.

Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

ß-diketone accumulation in response to drought stress is weakened in modern bread wheat varieties (Triticum aestivum L.).

Cuticular waxes coating leaf surfaces can help plants tolerate drought events by reducing non-stomatal water loss. Despite their role in drought tolerance, little is known about how cuticular wax composition has changed during breeding in Canadian bread wheat (Triticum aestivum L.) varieties. To fill in this gap, flag leaves of the Canadian Heritage Bread Wheat Panel, which include 30 varieties released between 1842 and 2018, were surveyed to determine if and how cuticular wax composition in wheat has changed at two breeding ecozones over this period. Following this, a subset of varieties was subjected to drought conditions to compare their responses. As expected, modern varieties outperformed old varieties with a significantly larger head length and reaching maturity earlier. Yet, when challenged with drought, old varieties were able to significantly increase the accumulation of ß-diketones to a higher extent than modern varieties. Furthermore, RNAseq was performed on the flag leaf of four modern varieties to identify potential markers that could be used for selection of higher accumulation of cuticular waxes. This analysis revealed that the W1 locus is a good candidate for selecting higher accumulation of ß-diketones. These findings indicate that the variation in cuticular waxes upon drought could be further incorporated in breeding of future bread wheat varieties.

Evolution of TPS20-related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites.

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono- and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS-e/f subfamily that utilize the unusual cis-prenyl diphosphate substrates neryl diphosphate and 2z,6z-farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS-e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α-pinene and limonene, or the sesquiterpene 7-epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra-specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.

Striking natural diversity in glandular trichome acylsugar composition is shaped by variation at the Acyltransferase2 locus in the wild tomato Solanum habrochaites.

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.

Tomato defences modulate not only insect performance but also their gut microbial composition.

Plants protect their tissues from insect herbivory with specialized structures and chemicals, such as cuticles, trichomes, and metabolites contained therein. Bacteria inside the insect gut are also exposed to plant defences and can potentially modify the outcome of plant-insect interactions. To disentangle this complex multi-organism system, we used tomato mutants impaired in the production of plant defences (odorless-2 and jasmonic acid-insensitive1) and two cultivars (Ailsa Craig and Castlemart), exposed them to herbivory by the cabbage looper (Trichoplusia ni H.) and collected the insect frass for bacterial community analysis. While the epicuticular wax and terpene profiles were variable, the leaf fatty acid composition remained consistent among genotypes. Moreover, larval weight confirmed the negative association between plant defences and insect performance. The distinctive frass fatty acid profiles indicated that plant genotype also influences the lipid digestive metabolism of insects. Additionally, comparisons of leaf and insect-gut bacterial communities revealed a limited overlap in bacterial species between the two sample types. Insect bacterial community abundance and diversity were notably reduced in insects fed on the mutants, with Enterobacteriaceae being the predominant group, whereas putatively pathogenic taxa were found in wildtype genotypes. Altogether, these results indicate that plant defences can modulate insect-associated bacterial community composition.

Chemical Pulping Advantages of Zip-lignin Hybrid Poplar.

Hybrid poplar genetically engineered to possess chemically labile ester linkages in its lignin backbone (zip-lignin hybrid poplar) was examined to determine if the strategic lignin modifications would enhance chemical pulping efficiencies. Kraft pulping of zip-lignin and wild-type hybrid poplar was performed in lab-scale reactors under conditions of varying severity by altering time, temperature and chemical charge. The resulting pulps were analyzed for yield, residual lignin content, and cellulose DP (degree of polymerization), as well as changes in carbohydrates and lignin structure. Statistical models of pulping were created, and the pulp bleaching and physical properties evaluated. Under identical cooking conditions, compared to wild-type, the zip-lignin hybrid poplar showed extended delignification, confirming the zip-lignin effect. Additionally, yield and carbohydrate content of the ensuing pulps were slightly elevated, as was the cellulose DP for zip-lignin poplar pulp, although differences in residual lignin between zip-lignin and wild-type poplar were not detected. Statistical prediction models facilitated comparisons between pulping conditions that resulted in identical delignification, with the zip-lignin poplar needing milder cooking conditions and resulting in higher pulp yield (up to 1.41 % gain). Bleaching and physical properties were subsequently equivalent between the samples with slight chemical savings realized in the zip-lignin samples due to the enhanced delignification.

Stability of plant defense proteins in the gut of insect herbivores.

Plant defense against insect herbivores is mediated in part by enzymes that impair digestive processes in the insect gut. Little is known about the evolutionary origins of these enzymes, their distribution in the plant kingdom, or the mechanisms by which they act in the protease-rich environment of the animal digestive tract. One example of such an enzyme is threonine (Thr) deaminase (TD), which in tomato (Solanum lycopersicum) serves a dual role in isoleucine (Ile) biosynthesis in planta and Thr degradation in the insect midgut. Here, we report that tomato uses different TD isozymes to perform these functions. Whereas the constitutively expressed TD1 has a housekeeping role in Ile biosynthesis, expression of TD2 in leaves is activated by the jasmonate signaling pathway in response to herbivore attack. Ingestion of tomato foliage by specialist (Manduca sexta) and generalist (Trichoplusia ni) insect herbivores triggered proteolytic removal of TD2's C-terminal regulatory domain, resulting in an enzyme that degrades Thr without being inhibited through feedback by Ile. This processed form (pTD2) of TD2 accumulated to high levels in the insect midgut and feces (frass). Purified pTD2 exhibited biochemical properties that are consistent with a postingestive role in defense. Shotgun proteomic analysis of frass from tomato-reared M. sexta identified pTD2 as one of the most abundant proteins in the excrement. Among the other tomato proteins identified were several jasmonate-inducible proteins that have a known or proposed role in anti-insect defense. Subtilisin-like proteases and other pathogenesis-related proteins, as well as proteins of unknown function, were also cataloged. We conclude that proteomic analysis of frass from insect herbivores provides a robust experimental approach to identify hyperstable plant proteins that serve important roles in defense.