Oxidosqualene cyclases involved in the biosynthesis of triterpenoids in Quercus suber cork

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (\u3e85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork (QsOSC1-3). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1-3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis

Silencing of StKCS6 in potato periderm leads to reduced chain lengths of suberin and wax compounds and increased peridermal transpiration

Very long chain aliphatic compounds occur in the suberin polymer and associated wax. Up to now only few genes involved in suberin biosynthesis have been identified. This is a report on the isolation of a potato (Solanum tuberosum) 3-ketoacyl-CoA synthase (KCS) gene and the study of its molecular and physiological relevance by means of a reverse genetic approach. This gene, called StKCS6, was stably silenced by RNA interference (RNAi) in potato. Analysis of the chemical composition of silenced potato tuber periderms indicated that StKCS6 down-regulation has a significant and fairly specific effect on the chain length distribution of very long-chain fatty acids (VLCFAs) and derivatives, occurring in the suberin polymer and peridermal wax. All compounds with chain lengths of C28 and higher were significantly reduced in silenced periderms, whereas compounds with chain lengths of C26 and lower accumulated. Thus, StKCS6 is preferentially involved in the formation of suberin and wax lipidic monomers with chain lengths of C28 and higher. As a result, peridermal transpiration of the silenced lines was about 1.5-times higher than that of the wild type. Our results convincingly show that StKCS6 is involved in both suberin and wax biosynthesis and that a reduction of the monomeric carbon chain lengths leads to increased rates of peridermal transpiration

Testicular spermatozoa and male germ cells evolution in "Dina lineata" (Hirudinea: Erpobdellidae) studied by scanning electron microscopy

A Dina lineata, hirudini erpobdèllid amb fecundació traumàtica hipodèrmica, cada espermatogònia primitiva, lliure en els sacs testiculars, entra en un procés de set divisions mitòtiques anticlinals cariocinètiques i no citocinètiques per formar poliplasts amb 2, 4, 8, 16, 32, 64 espermatogònies i, finalment, 128 espermatòcits primaris. Les cellules, en un mateix poliplast, evolucionen sincrònicament lligades per un pont a una massa citoplasmàtica central comuna anomenada citòfor. Una darrera divisió, meiòtica, porta a un poliplast amb 512 espermàtides. Per un complex procés morfogenètic de diferenciació cellular, cada espermàtida, arrodonida i d'uns 1,3 µm de diàmetre, esdevé un espermatozoide filamentós i helicoïdal d'uns 48 µm de longitud per 0,3 µm de diàmetre. L'ordre cronològic de diferenciació de les diverses regions del gàmeta és el següent: elongació del flagel, elongació i espiralització del nucli, elongació de la regió mitocondrial i elongació i espiralització, primer de l'acrosoma posterior i, després, de l'acrosoma anterior. A l'espermatozoide: 1) L'acrosoma posterior (7 µm) és una doble hèlix dextrògira formada per dues fibres, una d'estreta i una altra d'ampla, que descriuen, cadascuna, set voltes completes. 2) L'acrosoma anterior (6,5 µm) és un eix a l'entorn del qual una mateixa expansió cintiforme descriu, en sentit dextrògir, set voltes completes. 3) La regió nuclear (5,5 µm) és també una doble hèlix dextrògira integrada per dues fibres. A la base presenten el mateix gruix, però, a mesura que se n'allunyen, l'alteren a voltes alternes. 4) Les regions mitocondrial (8,5 µm) i flagellar (20 µm) són externament llises.This paper studies the male germ cells evolution in the leech Dina lineata by scanning electron microscopy. A primitive spermatogonium is an oval cell measuring 1,6 per 6 µm. Seven not-finished consecutive anticlinal mitotic divisions lead to the formation of isogenic groups with 2, 4, 8, 16, 32, 64 spermatogonia and, finally, 128 primary spermatocytes. The cells are all clustered in only one group and they evolve gradually and synchronically, remaining connected by cytoplasmic bridges to a central cytoplasmic mass called cytophore. A last division, which is meiotic, gives rise to an isogenic group with 512 spermatids: the cellular body of each one is reduced to 1,3 µm in diameter. By means of a complex spermiogenic process of cellular differentiation, each body becomes a filiform and helicoid spermatozoon measuring 48 µm in length and 0.3 µm in diameter. The morphogenetic and chronologic sequence of the various regions is the following: flagellum elongation; nucleus spiralization and elongation; mitochondrial region elongation; and finally, spiralization and elongation, first of the posterior acrosome and after of the anterior acrosome. In the spermatozoon: 1) The posterior acrosome (7 µm) is a right-handed double helix integrated by two fibers: a narrow one and a wide one; each describes seven complete turns. 2) The anterior acrosome (6.5 µm) is a longitudinal axis which has a ribbonshaped expansion describing seven right-handed complete turns around the same axis. 3) The nuclear region (5.5 µm) is also a right-handed double helix integrated by two fibers. On their base they show the same trickness, but this is alternatively altered along it from the base to the other side. 4) The mitochondrial region (8.5 µm) and the flagellar one (20 µm) are externally smooth

Oxidosqualene cyclases involved in the biosynthesis of triterpenoids in Quercus suber cork

Cork is a water-impermeable, suberin-based material harboring lignin, (hemi)cellulose, and extractable small molecules (primarily triterpenoids). Extractables strongly influence the properties of suberin-based materials. Though these previous findings suggest a key role for triterpenoids in cork material quality, directly testing this idea is hindered in part because it is not known which genes control cork triterpenoid biosynthesis. Here, we used gas chromatography and mass spectrometry to determine that the majority (\u3e85%) of non-polar extractables from cork were pentacyclic triterpenoids, primarily betulinic acid, friedelin, and hydroxy-friedelin. In other plants, triterpenoids are generated by oxidosqualene cyclases (OSCs). Accordingly, we mined Quercus suber EST libraries for OSC fragments to use in a RACE PCR-based approach and cloned three full-length OSC transcripts from cork (QsOSC1-3). Heterologous expression in Saccharomyces cerevisiae revealed that QsOSC1-3 respectively encoded enzymes with lupeol synthase, mixed α- and β-amyrin synthase, and mixed β-amyrin and friedelin synthase activities. These activities together account for the backbone structures of the major cork triterpenoids. Finally, we analyzed the sequences of QsOSC1-3 and other plant OSCs to identify residues associated with specific OSC activities, then combined this with analyses of Q. suber transcriptomic and genomic data to evaluate potential redundancies in cork triterpenoid biosynthesis

A Genomic Approach to Suberin Biosynthesis and Cork Differentiation1[C][W][OA]

Cork (phellem) is a multilayered dead tissue protecting plant mature stems and roots and plant healing tissues from water loss and injuries. Cork cells are made impervious by the deposition of suberin onto cell walls. Although suberin deposition and cork formation are essential for survival of land plants, molecular studies have rarely been conducted on this tissue. Here, we address this question by combining suppression subtractive hybridization together with cDNA microarrays, using as a model the external bark of the cork tree (Quercus suber), from which bottle cork is obtained. A suppression subtractive hybridization library from cork tree bark was prepared containing 236 independent sequences; 69% showed significant homology to database sequences and they corresponded to 135 unique genes. Out of these genes, 43.5% were classified as the main pathways needed for cork biosynthesis. Furthermore, 19% could be related to regulatory functions. To identify genes more specifically required for suberin biosynthesis, cork expressed sequence tags were printed on a microarray and subsequently used to compare cork (phellem) to a non-suberin-producing tissue such as wood (xylem). Based on the results, a list of candidate genes relevant for cork was obtained. This list includes genes for the synthesis, transport, and polymerization of suberin monomers such as components of the fatty acid elongase complexes, ATP-binding cassette transporters, and acyltransferases, among others. Moreover, a number of regulatory genes induced in cork have been identified, including MYB, No-Apical-Meristem, and WRKY transcription factors with putative functions in meristem identity and cork differentiation

Deconstructing a Plant Macromolecular Assembly: Chemical Architecture, Molecular Flexibility, And Mechanical Performance of Natural and Engineered Potato Suberins

Periderms present in plant barks are essential protective barriers to water diffusion, mechanical breakdown, and pathogenic invasion. They consist of densely packed layers of dead cells with cell walls that are embedded with suberin. Understanding the interplay of molecular structure, dynamics, and biomechanics in these cell wall-associated insoluble amorphous polymeric assemblies presents substantial investigative challenges. We report solid-state NMR coordinated with FT-IR and tensile strength measurements for periderms from native and wound-healing potatoes and from potatoes with genetically modified suberins. The analyses include the intact suberin aromatic–aliphatic polymer and cell-wall polysaccharides, previously reported soluble depolymerized transmethylation products, and undegraded residues including suberan. Wound-healing suberized potato cell walls, which are 2 orders of magnitude more permeable to water than native periderms, display a strikingly enhanced hydrophilic–hydrophobic balance, a degradation-resistant aromatic domain, and flexibility suggestive of an altered supramolecular organization in the periderm. Suppression of ferulate ester formation in suberin and associated wax remodels the periderm with more flexible aliphatic chains and abundant aromatic constituents that can resist transesterification, attenuates cooperative hydroxyfatty acid motions, and produces a mechanically compromised and highly water-permeable periderm

Potato native and wound periderms are differently affected by down-regulation of FHT, a suberin feruloyl transferase

Potato native and wound healing periderms contain an external multilayered phellem tissue (potato skin) consisting of dead cells whose cell walls are impregnated with suberin polymers. The phellem provides physical and chemical barriers to tuber dehydration, heat transfer, and pathogenic infection. Previous RNAi-mediated gene silencing studies in native periderm have demonstrated a role for a feruloyl transferase (FHT) in suberin biosynthesis and revealed how its down-regulation affects both chemical composition and physiology. To complement these prior analyses and to investigate the impact of FHT deficiency in wound periderms, a bottom-up methodology has been used to analyze soluble tissue extracts and solid polymers concurrently. Multivariate statistical analysis of LC-MS and GC-MS data, augmented by solid-state NMR and thioacidolysis, yields two types of new insights: the chemical compounds responsible for contrasting metabolic profiles of native and wound periderms, and the impact of FHT deficiency in each of these plant tissues. In the current report, we confirm a role for FHT in developing wound periderm and highlight its distinctive features as compared to the corresponding native potato periderm.This work was supported by grants from the U.S. National Science Foundation (NSF MCB-0843627, 1411984, and 0741914 to R.E.S.), from the Spanish Ministerio de Economía y Competitividad and FEDER funding (AGL2012-36725; AGL2015-67495-C2-1-R), and from the University of Guelph (MPCUdG2016/078). The NMR resources were operated by The City College of New York and the CUNY Institute for Macromolecular Assemblies. Infrastructural support for the NMR facilities was provided by the U.S. National Institutes of Health (5G12MD007603-30 from the National Institute on Minority Health and Health Disparities). The GC-MS instrument was supported by the U.S. NSF (CHE-0840498) and the GC-FID instrument by the University of Guelph (SING11/1).Peer reviewe