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.

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.

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.

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.

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.

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.

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.

A comparative transcriptomic approach to understanding the formation of cork.

KEY MESSAGE: The transcriptome comparison of two oak species reveals possible candidates accounting for the exceptionally thick and pure cork oak phellem, such as those involved in secondary metabolism and phellogen activity. Cork oak, Quercus suber, differs from other Mediterranean oaks such as holm oak (Quercus ilex) by the thickness and organization of the external bark. While holm oak outer bark contains sequential periderms interspersed with dead secondary phloem (rhytidome), the cork oak outer bark only contains thick layers of phellem (cork rings) that accumulate until reaching a thickness that allows industrial uses. Here we compare the cork oak outer bark transcriptome with that of holm oak. Both transcriptomes present similitudes in their complexity, but whereas cork oak external bark is enriched with upregulated genes related to suberin, which is the main polymer responsible for the protective function of periderm, the upregulated categories of holm oak are enriched in abiotic stress and chromatin assembly. Concomitantly with the upregulation of suberin-related genes, there is also induction of regulatory and meristematic genes, whose predicted activities agree with the increased number of phellem layers found in the cork oak sample. Further transcript profiling among different cork oak tissues and conditions suggests that cork and wood share many regulatory mechanisms, probably reflecting similar ontogeny. Moreover, the analysis of transcripts accumulation during the cork growth season showed that most regulatory genes are upregulated early in the season when the cork cambium becomes active. Altogether our work provides the first transcriptome comparison between cork oak and holm oak outer bark, which unveils new regulatory candidate genes of phellem development.

The Identification and Quantification of Suberin Monomers of Root and Tuber Periderm from Potato (Solanum tuberosum) as Fatty Acyl tert-Butyldimethylsilyl Derivatives.

INTRODUCTION: Protective plant lipophilic barriers such as suberin and cutin, with their associated waxes, are complex fatty acyl derived polyesters. Their precise chemical composition is valuable to understand the specific role of each compound to the physiological function of the barrier. OBJECTIVES: To develop a method for the compositional analysis of suberin and associated waxes by gas chromatography (GC) coupled to ion trap-mass spectrometry (IT-MS) using N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) as sylilating reagent, and apply it to compare the suberin of the root and tuber periderm of potato (Solanum tuberosum). METHODOLOGY: Waxes and suberin monomers from root and periderm were extracted subsequently using organic solvents and by methanolysis, and subjected to MTBSTFA derivatisation. GC analyses of periderm extracts were used to optimise the chromatographic method and the compound identification. Quantitative data was obtained using external calibration curves. The method was fully validated and applied for suberin composition analyses of roots and periderm. RESULTS: Wax and suberin compounds were successfully separated and compound identification was based on the specific (M-57) and non-specific ions in mass spectra. The use of calibration curves built with different external standards provided quantitative accurate data and showed that suberin from root contains shorter chained fatty acyl derivatives and a relative predominance of α,ω-alkanedioic acids compared to that of the periderm. CONCLUSION: We present a method for the analysis of suberin and their associated waxes based on MTBSTFA derivatisation. Moreover, the characteristic root suberin composition may be the adaptive response to its specific regulation of permeability to water and gases. Copyright © 2016 John Wiley & Sons, Ltd.

Silencing of the potato StNAC103 gene enhances the accumulation of suberin polyester and associated wax in tuber skin.

Suberin and wax deposited in the cork (phellem) layer of the periderm form the lipophilic barrier that protects mature plant organs. Periderm lipids have been widely studied for their protective function with regards to dehydration and for how they respond to environmental stresses and wounding. However, despite advances in the biosynthetic pathways of suberin and associated wax, little is known about the regulation of their deposition. Here, we report on a potato NAC transcription factor gene, StNAC103, induced in the tuber phellem (skin). The StNAC103 promoter is active in cells undergoing suberization such as in the basal layer of the phellem, but also in the root apical meristem. Gene silencing in potato periderm correlates with an increase in the suberin and wax load, and specifically in alkanes, ω-hydroxyacids, diacids, ferulic acid, and primary alcohols. Concomitantly, silenced lines also showed up-regulation of key genes related to the biosynthesis and transport of suberin and wax in the tuber periderm. Taken together, our results suggest that StNAC103 has a role in the tight regulation of the formation of apoplastic barriers and is, to the best of our knowledge, the first candidate gene to be identified as being involved in the repression of suberin and wax deposition.

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.

The potato suberin feruloyl transferase FHT which accumulates in the phellogen is induced by wounding and regulated by abscisic and salicylic acids.

The present study provides new insights on the role of the potato (Solanum tuberosum) suberin feruloyl transferase FHT in native and wound tissues, leading to conclusions about hitherto unknown properties of the phellogen. In agreement with the enzymatic role of FHT, it is shown that its transcriptional activation and protein accumulation are specific to tissues that undergo suberization such as the root boundary layers of the exodermis and the endodermis, along with the tuber periderm. Remarkably, FHT expression and protein accumulation within the periderm is restricted to the phellogen derivative cells with phellem identity. FHT levels in the periderm are at their peak near harvest during periderm maturation, with the phellogen becoming meristematically inactive and declining thereafter. However, periderm FHT levels remain high for several months after harvest, suggesting that the inactive phellogen retains the capacity to synthesize ferulate esters. Tissue wounding induces FHT expression and the protein accumulates from the first stages of the healing process onwards. FHT is up-regulated by abscisic acid and down-regulated by salicylic acid, emphasizing the complex regulation of suberin synthesis and wound healing. These findings open up new prospects important for the clarification of the suberization process and yield important information with regard to the skin quality of potatoes.

A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation.

Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

Unraveling ferulate role in suberin and periderm biology by reverse genetics.

Plant cell walls are dramatically affected by suberin deposition, becoming an impermeable barrier to water and pathogens. Suberin is a complex layered heteropolymer that comprises both a poly(aliphatic) and a poly(aromatic) lignin-like domain. Current structural models for suberin attribute the crosslinking of aliphatic and aromatic domains within the typical lamellar ultrastructure of the polymer to esterified ferulate. BAHD feruloyl transferases involved in suberin biosynthesis have been recently characterized in Arabidopsis and potato (Solanum tuberosum). In defective mutants, suberin, even lacks most of the esterified ferulate, but maintains the typical lamellar ultrastructure. However, suberized tissues display increased water permeability, in spite of exhibiting a similar lipid load to wild type. Therefore, the role of ferulate in suberin needs to be reconsidered. Moreover, silencing the feruloyl transferase in potato turns the typical smooth skin of cv. Desirée into a rough scabbed skin distinctive of Russet varieties and impairs the normal skin maturation that confers resistance to skinning. Concomitantly to these changes, the skin of silenced potatoes shows an altered profile of soluble phenolics with the emergence of conjugated polyamines.