Rhytidome- and cork-type barks of holm oak, cork oak and their hybrids highlight processes leading to cork formation.

BACKGROUND: The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. In most trees, like holm oak, the first periderm is frequently replaced by subsequent internal periderms yielding a heterogeneous outer bark made of a mixture of periderms and phloem tissues, known as rhytidome. Exceptionally, cork oak forms a persistent or long-lived periderm which results in a homogeneous outer bark of thick phellem cell layers known as cork. Cork oak and holm oak distribution ranges overlap to a great extent, and they often share stands, where they can hybridize and produce offspring showing a rhytidome-type bark. RESULTS: Here we use the outer bark of cork oak, holm oak, and their natural hybrids to analyse the chemical composition, the anatomy and the transcriptome, and further understand the mechanisms underlying periderm development. We also include a unique natural hybrid individual corresponding to a backcross with cork oak that, interestingly, shows a cork-type bark. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach cork and rhytidome development, allowing an accurate identification of candidate genes and processes. The present study underscores that abiotic stress and cell death are enhanced in rhytidome-type barks whereas lipid metabolism and cell cycle are enriched in cork-type barks. Development-related DEGs showing the highest expression, highlight cell division, cell expansion, and cell differentiation as key processes leading to cork or rhytidome-type barks. CONCLUSION: Transcriptome results, in agreement with anatomical and chemical analyses, show that rhytidome and cork-type barks are active in periderm development, and suberin and lignin deposition. Development and cell wall-related DEGs suggest that cell division and expansion are upregulated in cork-type barks whereas cell differentiation is enhanced in rhytidome-type barks.

Genetic Basis of Breast and Ovarian Cancer: Approaches and Lessons Learnt from Three Decades of Inherited Predisposition Testing.

Germline variants occurring in BRCA1 and BRCA2 give rise to hereditary breast and ovarian cancer (HBOC) syndrome, predisposing to breast, ovarian, fallopian tube, and peritoneal cancers marked by elevated incidences of genomic aberrations that correspond to poor prognoses. These genes are in fact involved in genetic integrity, particularly in the process of homologous recombination (HR) DNA repair, a high-fidelity repair system for mending DNA double-strand breaks. In addition to its implication in HBOC pathogenesis, the impairment of HR has become a prime target for therapeutic intervention utilizing poly (ADP-ribose) polymerase (PARP) inhibitors. In the present review, we introduce the molecular roles of HR orchestrated by BRCA1 and BRCA2 within the framework of sensitivity to PARP inhibitors. We examine the genetic architecture underneath breast and ovarian cancer ranging from high- and mid- to low-penetrant predisposing genes and taking into account both germline and somatic variations. Finally, we consider higher levels of complexity of the genomic landscape such as polygenic risk scores and other approaches aiming to optimize therapeutic and preventive strategies for breast and ovarian cancer.

Randomized, open-label, phase II, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with locally advanced, inflammatory, or early HER2-positive breast cancer-Immun-HER trial (GOIRC-01-2016).

BACKGROUND: It is possible to induce immunomodulation in HER2-positive breast cancer (BC) by modifying the route of administration of trastuzumab. METHODS: In this multicenter randomized phase II trial, all enrolled patients (pts) with T2-T4d HER2-positive BC received 3 cycles of neoadjuvant treatment (NAT) with fluorouracil, epirubicin and cyclophosphamide every 3 weeks (q21), followed by docetaxel/pertuzumab plus intravenous trastuzumab (arm A) or, docetaxel/pertuzumab plus subcutaneous (SC) trastuzumab (arm B) q21x4 cycles. After surgical operation, each pt was treated with trastuzumab q21x14 cycles using the same SC or intravenous formulation of NAT. Primary endpoint was the proportion of subjects with high stromal tumor-infiltrating lymphocytes (sTILs) in postneoadjuvant residual disease (RD). RESULTS: Sixty-three pts (31 (arm A) and 32 (arm B)) were enrolled. Pathological complete response was obtained by 20/31 pts (64.5%; 95% CI 45.4% to 80.1%) in arm A and 19/32 pts (59.4%; 95% CI 40.1% to 76.3%) in arm B. High sTILs were observed in 27% and 46% of postneoadjuvant residual tumors in arms A and B, respectively. CD8+ T cells increased significantly in RDs of both arms (p=0.014 and 0.002 for arm A and B, respectively), whereas a significant decline in the level of CD4+ FoxP3+ regulatory T cells was observed only in arm B (p=0.016). A significant upregulation of PD-1 on sTILs was found in RD of pts enrolled in arm B (p=0.012), while programmed death-ligand 1 (PD-L1) was significantly overexpressed in residual tumors of arm A (p=0.02). A strong negative correlation was reported in arm B between expression of PD-L1 on pretreatment sTILs and CD3 expression on sTILs in RD (τ: -0.73). Grade≥3 AE incidence rates were similar between the two arms. CONCLUSIONS: SC trastuzumab induced relevant sTILs enrichment, with favorable variations of immune parameters in HER2-positive BC pts with RD after NAT. Novel immunotherapy strategies should be tested to achieve SC-specific, antitumor immune response. TRIAL REGISTRATION NUMBER: NCT03144947, and EudraCT number: 2016-000435-41.

The Tomato Feruloyl Transferase FHT Promoter Is an Accurate Identifier of Early Development and Stress-Induced Suberization.

As a wall polymer, suberin has a multifaceted role in plant development and stress responses. It is deposited between the plasma membrane and the primary cell wall in specialized tissues such as root exodermis, endodermis, phellem, and seed coats. It is formed de novo in response to stresses such as wounding, salt injury, drought, and pathogen attack and is a complex polyester mainly consisting of fatty acids, glycerol, and minor amounts of ferulic acid that are associated to a lignin-like polymer predominantly composed of ferulates. Metabolomic and transcriptomic studies have revealed that cell wall lignification precedes suberin deposition. The ferulic acid esterified to ω-hydroxy fatty acids, synthetized by the feruloyl transferase FHT (or ASFT), presumably plays a role in coupling both polymers, although the precise mechanism is not understood. Here, we use the promoter of tomato suberin feruloyl transferase (FHT/ASFT) fused to GUS (ß-glucuronidase) to demonstrate that ferulate deposition agrees with the site of promoter FHT activation by using a combination of histochemical staining and UV microscopy. Hence, FHT promoter activation and alkali UV microscopy can be used to identify the precise localization of early suberizing cells rich in ferulic acid and can additionally be used as an efficient marker of early suberization events during plant development and stress responses. This line can be used in the future as a tool to identify emerging suberization sites via ferulate deposition in tomato plants, which may contribute to germplasm screening in varietal improvement programs.

The making of suberin.

Outer protective barriers of animals use a variety of bio-polymers, based on either proteins (e.g. collagens), or modified sugars (e.g. chitin). Plants, however, have come up with a particular solution, based on the polymerisation of lipid-like precursors, giving rise to cutin and suberin. Suberin is a structural lipophilic polyester of fatty acids, glycerol and some aromatics found in cell walls of phellem, endodermis, exodermis, wound tissues, abscission zones, bundle sheath and other tissues. It deposits as a hydrophobic layer between the (ligno)cellulosic primary cell wall and plasma membrane. Suberin is highly protective against biotic and abiotic stresses, shows great developmental plasticity and its chemically recalcitrant nature might assist the sequestration of atmospheric carbon by plants. The aim of this review is to integrate the rapidly accelerating genetic and cell biological discoveries of recent years with the important chemical and structural contributions obtained from very diverse organisms and tissue layers. We critically discuss the order and localisation of the enzymatic machinery synthesising the presumed substrates for export and apoplastic polymerisation. We attempt to explain observed suberin linkages by diverse enzyme activities and discuss the spatiotemporal relationship of suberin with lignin and ferulates, necessary to produce a functional suberised cell wall.

Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato.

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

The Making of Plant Armor: The Periderm.

The periderm acts as armor protecting the plant's inner tissues from biotic and abiotic stress. It forms during the radial thickening of plant organs such as stems and roots and replaces the function of primary protective tissues such as the epidermis and the endodermis. A wound periderm also forms to heal and protect injured tissues. The periderm comprises a meristematic tissue called the phellogen, or cork cambium, and its derivatives: the lignosuberized phellem and the phelloderm. Research on the periderm has mainly focused on the chemical composition of the phellem due to its relevance as a raw material for industrial processes. Today, there is increasing interest in the regulatory network underlying periderm development as a novel breeding trait to improve plant resilience and to sequester CO2. Here, we discuss our current understanding of periderm formation, focusing on aspects of periderm evolution, mechanisms of periderm ontogenesis, regulatory networks underlying phellogen initiation and cork differentiation, and future challenges of periderm research.

Silencing of StRIK in potato suggests a role in periderm related to RNA processing and stress.

BACKGROUND: The periderm is a protective barrier crucial for land plant survival, but little is known about genetic factors involved in its development and regulation. Using a transcriptomic approach in the cork oak (Q. suber) periderm, we previously identified an RS2-INTERACTING KH PROTEIN (RIK) homologue of unknown function containing a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. RESULTS: To gain insight into the function of RIK in the periderm, potato (S. tuberosum) tuber periderm was used as a model: the full-length coding sequence of RIK, hereafter referred to as StRIK, was isolated, the transcript profile analyzed and gene silencing in potato performed to analyze the silencing effects on periderm anatomy and transcriptome. The StRIK transcript accumulated in all vegetative tissues studied, including periderm and other suberized tissues such as root and also in wounded tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show any obvious effects on tuber periderm anatomy but, unlike Wild type, transgenic plants flowered. Global transcript profiling of the StRIK-RNAi periderm did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis orthologue. CONCLUSIONS: The ubiquitous expression of StRIK transcript, the flower associated phenotype and the differential expression of StRIK-RNAi periderm point out to a general regulatory role of StRIK in diverse plant developmental processes. The transcriptome analysis suggests that StRIK might play roles in RNA maturation and stress response in the periderm.

A chemical window into the impact of RNAi silencing of the StNAC103 gene in potato tuber periderms: Soluble metabolites, suberized cell walls, and antibacterial defense.

The growth and survival of terrestrial plants require control of their interactions with the environment, e.g., to defend against desiccation and microbial invasion. For major food crops, the protection conferred by the outer skins (periderm in potato) is essential to cultivation, storage, and marketing of the edible tubers and fruits. Potatoes are particularly vulnerable to bacterial infections due to their high content of water and susceptibility to mechanical wounding. Recently, both specific and conserved gene silencing (StNAC103-RNAi and StNAC103-RNAi-c, respectively) were found to increase the load of wax and aliphatic suberin depolymerization products in tuber periderm, implicating this NAC gene as a repressor of the wax and suberin biosynthetic pathways. However, an important gap in our understanding of StNAC103 silencing concerns the metabolites produced in periderm cells as antimicrobial defense agents and potential building blocks of the deposited suberin biopolymer. In the current work, we have expanded prior studies on StNAC103 silenced lines by conducting comprehensive parallel analyses to profile changes in chemical constituents and antibacterial activity. Compositional analysis of the intact suberized cell walls using solid-state 13C NMR (ssNMR) showed that NAC silencing produced an increase in the long-chain aliphatic groups deposited within the periderm cell walls. LC-MS of polar extracts revealed up-regulation of glycoalkaloids in both StNAC103-RNAi and StNAC103-RNAi-c native periderms but down-regulation of a phenolic amine in StNAC103-RNAi-c and a phenolic acid in StNAC103-RNAi native periderms. The nonpolar soluble metabolites identified using GC-MS included notably abundant long-chain alkane metabolites in both silenced samples. By coordinating the differentially accumulated soluble metabolites and the suberin depolymerization products with the ssNMR-based profiles for the periderm polymers, it was possible to obtain a holistic view of the chemical changes that result from StNAC103 gene silencing. Correspondingly, the chemical composition trends served as a backdrop to interpret trends in the chemical barrier defense function of native tuber periderms, which was found to be more robust for the nonpolar extracts.

Gene Downregulation in Potato Roots Using Agrobacterium rhizogenes-Mediated Transformation.

Agrobacterium rhizogenes has the ability to transform plant cells by transferring the T-DNA from the Ri plasmid to the plant cell genome. These infected plant cells divide and organize the formation of adventitious roots, called hairy roots. When the A. rhizogenes is additionally transformed with a binary vector, the cells infected can indeed be transformed with this second T-DNA producing transgenic hairy roots. In this chapter, we present the protocol to produce transgenic hairy roots from in vitro potato (Solanum tuberosum) plants injected with transformed A. rhizogenes, generating plants with a wild-type shoot and a transgenic root system. Specifically, we detail the procedure to obtain in vitro-cultured hairy roots with a downregulated gene of interest, by using a Gateway-based binary vector able to produce a RNA hairpin triggering the RNA interference mechanism (hpRNAi). We also present the protocol to analyze the downregulation of the target gene in hairy roots by means of reverse-transcription reaction followed by real-time PCR (qPCR).

Transcriptomic analysis of cork during seasonal growth highlights regulatory and developmental processes from phellogen to phellem formation.

The phellogen or cork cambium stem cells that divide periclinally and outwardly specify phellem or cork. Despite the vital importance of phellem in protecting the radially-growing plant organs and wounded tissues, practically only the suberin biosynthetic process has been studied molecularly so far. Since cork oak (Quercus suber) phellogen is seasonally activated and its proliferation and specification to phellem cells is a continuous developmental process, the differentially expressed genes during the cork seasonal growth served us to identify molecular processes embracing from phellogen to mature differentiated phellem cell. At the beginning of cork growth (April), cell cycle regulation, meristem proliferation and maintenance and processes triggering cell differentiation were upregulated, showing an enrichment of phellogenic cells from which phellem cells are specified. Instead, at maximum (June) and advanced (July) cork growth, metabolic processes paralleling the phellem cell chemical composition, such as the biosynthesis of suberin, lignin, triterpenes and soluble aromatic compounds, were upregulated. Particularly in July, polysaccharides- and lignin-related secondary cell wall processes presented a maximal expression, indicating a cell wall reinforcement in the later stages of cork formation, presumably related with the initiation of latecork development. The putative function of relevant genes identified are discussed in the context of phellem ontogeny.

The AFLATOX® Project: Approaching the Development of New Generation, Natural-Based Compounds for the Containment of the Mycotoxigenic Phytopathogen Aspergillus flavus and Aflatoxin Contamination.

The control of the fungal contamination on crops is considered a priority by the sanitary authorities of an increasing number of countries, and this is also due to the fact that the geographic areas interested in mycotoxin outbreaks are widening. Among the different pre- and post-harvest strategies that may be applied to prevent fungal and/or aflatoxin contamination, fungicides still play a prominent role; however, despite of countless efforts, to date the problem of food and feed contamination remains unsolved, since the essential factors that affect aflatoxins production are various and hardly to handle as a whole. In this scenario, the exploitation of bioactive natural sources to obtain new agents presenting novel mechanisms of action may represent a successful strategy to minimize, at the same time, aflatoxin contamination and the use of toxic pesticides. The Aflatox® Project was aimed at the development of new-generation inhibitors of aflatoxigenic Aspergillus spp. proliferation and toxin production, through the modification of naturally occurring molecules: a panel of 177 compounds, belonging to the thiosemicarbazones class, have been synthesized and screened for their antifungal and anti-aflatoxigenic potential. The most effective compounds, selected as the best candidates as aflatoxin containment agents, were also evaluated in terms of cytotoxicity, genotoxicity and epi-genotoxicity to exclude potential harmful effect on the human health, the plants on which fungi grow and the whole ecosystem.

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 (>85%) 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 ß-amyrin synthase, and mixed ß-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 against the conserved NAC domain of the potato StNAC103 reveals new NAC candidates to repress the suberin associated waxes in phellem.

Both suberin and its associated waxes contribute to the formation of apoplastic barriers that protect plants from the environment. Some transcription factors have emerged as regulators of the suberization process. The potato StNAC103 gene was reported as a repressor of suberin polyester and suberin-associated waxes deposition because its RNAi-mediated downregulation (StNAC103-RNAi) over-accumulated suberin and associated waxes in the tuber phellem concomitantly with the induction of representative biosynthetic genes. Here, to explore if other genes of the large NAC gene family participate to this repressive function, we extended the silencing to other NAC members by targeting the conserved NAC domain of StNAC103 (StNAC103-RNAi-c). Transcript profile of the StNAC103-RNAi-c phellem indicated that StNAC101 gene was an additional potential target. In comparison with StNAC103-RNAi, the silencing with StNAC103-RNAi-c construct resulted in a similar effect in suberin but yielded an increased load of associated waxes in tuber phellem, mainly alkanes and feruloyl esters. Globally, the chemical effects in both silenced lines are supported by the transcript accumulation profile of genes involved in the biosynthesis, transport and regulation of apoplastic lipids. In contrast, the genes of polyamine biosynthesis were downregulated. Altogether these results point out to StNAC101 as a candidate to repress the suberin-associated waxes.

Antiaflatoxigenic Thiosemicarbazones as Crop-Protective Agents: A Cytotoxic and Genotoxic Study.

Aflatoxins are secondary fungal metabolites that can contaminate feed and food. They are a cause of growing concern worldwide, because they are potent carcinogenic agents. Thiosemicarbazones are molecules that possess interesting antiaflatoxigenic properties, but in order to use them as crop-protective agents, their cytotoxic and genotoxic profiles must first be assessed. In this paper, a group of thiosemicarbazones and a copper complex are reported as compounds able to antagonize aflatoxin biosynthesis, fungal growth, and sclerotia biogenesis in Aspergillus flavus. The two most interesting thiosemicarbazones found were noncytotoxic on several cell lines (CRL1790, Hs27, HFL1, and U937), and therefore, they were submitted to additional analysis of mutagenicity and genotoxicity on bacteria, plants, and human cells. No mutagenic activity was observed in bacteria, whereas genotoxic activity was revealed by the Alkaline Comet Assay on U937 cells and by the test of chromosomal aberrations in Allium cepa.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining.

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant's genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed in roots.

Use of FTA® classic cards for epigenetic analysis of sperm DNA.

FTA® technologies provide the most reliable method for DNA extraction. Although FTA technologies have been widely used for genetic analysis, there is no literature on their use for epigenetic analysis yet. We present for the first time, a simple method for quantitative methylation assessment based on sperm cells stored on Whatman FTA classic cards. Specifically, elution of seminal DNA from FTA classic cards was successfully tested with an elution buffer and an incubation step in a thermocycler. The eluted DNA was bisulfite converted, amplified by PCR, and a region of interest was pyrosequenced.

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.