Functional genomics of the periderm: the biosynthetic gene FHT, the transcriptional regulator StRiK and the transcriptome deciphering

We have developed new molecular tools to characterize the FHT and StRIK genes in the tuber periderm. Regarding FHT gene, our results demonstrated that is induced very specifically in suberizing tissues what makes FHT a good marker of the suberization process. Regarding StRIK gene, it has been shown to be a good candidate for the periderm regulation since its silencing causes changes in genes expression related to the transposition of DNA, RNA processing and stress. Finally, by RNA-seq we have identified a wide range of new candidate genes for the formation of the cork oak periderm. Among these genes several are related to the formation of the cell wall, cell primary metabolism and suberin accumulation. Other relevant genes are those involved in the regulation of meristem such as auxin transporters and the ethylene metabolism and signaling. The expression patterns of some genes have been studied during the cork growing season.Hem desenvolupat noves eines moleculars per a la caracterització dels gens FHT i StRIK en el peridermis del tubercle. Els nostres resultats mostren que FHT s’indueix de forma molt específica en teixits suberificats el que el fa un bon marcador del procés de suberificació. En relació al gen StRIK, hem vist que es un bon candidat a la regulació de la peridermis ja que el seu silenciament provoca canvis d’expressió en gens relacionats amb la transposició de l’ADN, el processament de l’RNA i l’estrès. Mitjançant RNA-seq hem identificat nous gens candidats per la formació de la peridermis en l’alzina surera, entre ells destaquen gens relacionats amb la formació de la paret cel·lular, el metabolisme primari i l’acumulació de suberina. També destaquen gens relacionats amb la regulació del meristema com ara els transportadors d’auxines i el metabolisme i senyalització per etilè. L’expressió d’alguns gens s’ha analitzat durant la formació del suro

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

Abstract 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

A comparative transcriptomic approach to understanding the formation of cork

International audienceCork 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 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 potatoesThe authors thank Professor S. Prat (Centro Nacional de Biotecnologia, Madrid) and Dr E. Dominguez (Centre de Recerca en Agrigenomica CRAG, Barcelona) for providing potato ssp. andigena, for support, and for helpful experimental advice; Dr P. Suarez (CRAG) for kindly lending us the ultracentrifuge; Professor D. Ludevid, Dr S. Irar, and Dr M. P. Gonzalez (CRAG), and Dr T. Roscoe (Institut de Recherche pour le Development, Montpellier) for fruitful suggestions regarding protein and antibody production, immunolocalization, and GUS staining; and Dr J. Castro (Biology Department, University of Girona, UdG) for helpful advice on setting up the western blot conditions. We also thank Mr J. Blavia and D. Reyes (Serveis Tecnics de Recerca, UdG) and S. Gomez (Departament de Biologia, UdG) for their valuable assistance in carrying out the laboratory work. This work was supported by the Ministerio de Innovacion y Ciencia [AGL2009-13745], the Ministerio de Educacion y Ciencia [FPI grant to PB], and the Ministerio de Economia y Competitividad [AGL2012-36725

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

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

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