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

Abstract

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.

Keywords: Cork; Cork oak; Holm oak; Hybrids; Outer bark; Periderm; Phellem; Rhytidome; Suberin.

Fig. 1

Outer bark anatomy of cork oak, holm oak and their hybrids. Suberized cell wall fluorescence detected in cross-sections under UV light after phloroglucinol-HCl staining. (A) Cork oak (Q. suber), (B) holm oak (Q. ilex), (C) F1 hybrid with rhytidome-like phenotype, (D) specific hybrid backcrossed with Q. suber and with a cork-type phenotype. Bright field (left) and UV (right) observations in each panel. Phellem layers (closed circle), suberized inactive phloem (open circle) and a lignified phloematic ray (closed square). Scale bars: 200 μm

Fig. 2

Chemical composition of the outer barks of cork oak, holm oak and their hybrids. (A) Principal component analysis (PCA) of the data from chemical composition analysis of the outer barks of cork oak, holm oak and the hybrids. The first principal component shows a clear separation between cork-type and rhytidome-type barks and a gradient between cork, cork-like hybrid and the rhytidome-type barks. (B) Dry weight % of the outer bark chemical composition of cork oak, holm oak, and a set of hybrids showing rhytidome-like bark and the hybrid showing a cork-like bark. Note the higher relative percentage of suberin and dichloromethane-soluble extractives in the cork-type barks

Fig. 3

Transcriptome profile and differential expression analysis of the different outer barks. (A) Principal component analysis of the global transcript profile obtained from the outer barks of cork oak, holm oak and the hybrids. Similar transcriptomes within individuals of the same bark-type group together. The first principal component shows a clear separation between cork-type and rhytidome-type barks, as well as a gradient between cork, cork-like and rhytidome-type outer bark. The second component separates the rhytidome and rhytidome-like outer bark at opposite ends. (B) Volcano plot showing odds of differential expression (-log10 p-adjusted value) against ratio (log2 FoldChange) of different pairwise comparisons: cork/rhytidome, cork/cork-like, cork/rhytidome-like, cork-like/rhytidome, rhytidome-like/rhytidome, cork-like/rhytidome-like. Genes with –log10 greater than 2 and with log2FC absolute value greater than 1 are considered as DEGs. Green dots depict upregulated genes and red dots downregulated genes for each comparative. The number of upregulated and downregulated genes found in each comparison are shown in green and red, respectively within each graph. (C) Correlation graph of the mRNAs log2FC values between the RNA-seq and the qPCR analyses. The Pearson correlation coefficient (ρ) is 0.804 and the p-value < 0.001 (3.43 10^− 9). The shaded area represents the confidence interval of the regression line

Fig. 4

Cluster analysis of DEGs according to their expression profile in the different outer bark types. Eight clusters were obtained. Each cluster panel shows the number of genes included and the individual and averaged gene expression profile (rlog) in grey and purple lines, respectively. Clusters 1, 2 and 3 contain genes upregulated in rhytidome-type outer barks. Clusters 4, 5, 6, and 8 contain genes upregulated in cork-type barks. Cluster 7 shows particular expression peaks in cork-like and rhytidome outer barks

Fig. 5

Summary of biological processes occurring during cork and rhytidome formation. This summary is based on upregulated genes and processes in cork-type and rhytidome-type outer barks from Q. suber, Q. ilex and their natural hybrids (cork-like and rhytidome-like). The outer tissue portion analysed corresponded to the inner face of the outer bark, which includes the meristematic active cells of phellogen and the alive phellem cells, and for rhytidome-type bark also included alive secondary phloem. Phellogen in Q. suber extends concentrically, is reactivated every growing season and forms a persistent periderm during the entire tree life called cork. In Q. ilex, the periderm is not persistent and is substituted for new and active phellogens formed inwardly within secondary phloem and yielding a rhytidome outer bark constituted by subsequent periderms with phloem tissue enclosed between them. The phelloderm, derived from each phellogen and located inwardly, has been omitted for simplicity; phelloderm, phellogen and phellem constitute each of the periderms depicted. Sketch inspired from Junikka [94]