Cork cells in cork oak periderms undergo programmed cell death and proanthocyanidin deposition

Abstract

Vascular plants with secondary growth develop a periderm mostly composed of dead suberized cork cells to face environmental hostile conditions. Cork oak has a highly active and long-living phellogen forming a remarkably thick periderm that is periodically debarked for industrial purposes. This wounding originates the quick formation of a new traumatic periderm, making cork oak an exceptional model to study the first periderm differentiation during normal development in young sprigs and traumatic (wound) periderm formation after debarking. Here we studied the poorly known first periderm differentiation steps that involve cell wall suberization, polyphenolic accumulation, and programmed cell death (PCD) by combining TEM, histochemical, and molecular methods in periderms from young sprigs. These processes were further compared with traumatic periderms formed after wounding using molecular and histochemical techniques, such as the polyphenolic accumulation. In the first periderms from young sprigs, four distinct differentiation stages were defined according to the presence of PCD morphological features. First young and traumatic periderms showed an upregulation of genes related to suberin biosynthesis, proanthocyanidins biosynthesis and transport, autophagy, and PCD. Traumatic periderms revealed an overall upregulation of these genes, likely resulting from ontogeny differences and distinct phellogen origin associated with a faster metabolism, highlighting the impact of wounding on phellogen activity after debarking. First periderms from young sprigs showed gradual accumulation of proanthocyanidins in the vacuoles throughout PCD stages until total filled lumens, whereas in traumatic periderms these compounds were found cell wall linked in already empty cells. This work enabled a comprehensive overview of the cork cells differentiation processes contributing to deepening the knowledge of the fundamental ontogenic program of this protective tissue, which is also a unique forest product, constituting the basis of a sustainable and profitable industry.

Spatiotemporal development of suberized barriers in cork oak taproots

“…These are polyphenolic secondary metabolites synthesized as oligomers or polymers via the flavonoid pathway (Sagasser et al 2002, Appelhagen et al 2010, 2011. We can hypothesize that the strongly expressed WIP3 is specially involved in the control of flavonoid and tannin accumulation, expected in the new cork oak phellem cells (Graça and Pereira 2004, Silva et al 2005, Inácio et al 2021, or other new secondary tissues (Crang et al 2018). Also in mature cork oak roots we detected the expression of TFs described as the first layer of regulation for secondary cell walls, such as the VND1/3/4 (LOC111996673/LOC111994497/LOC112007201, LOC111988979) and XND1 (LOC112009005) homologs (Zhang et al 2018).…”

Cork Development: What Lies Within

“…Further down the phenylpropanoid pathway acting in the early steps of the lignin branch, the enzyme hydroxycinnamoyltransferase (HCT) whose corresponding gene, when down-regulated, leads to a strong decrease of suberin-linked ferulic acid levels [ 77 ]. Proanthocyanidins (PA), the end product of the flavonoid pathway, a phenylpropanoid pathway branch [ 78 ], accumulates in the vacuoles of cells of the traumatic periderms and young cork cells, and associate with the cell walls of suberized empty cells [ 79 ]. Anatomy studies have already shown us that the first cork cells formed can be distinguished by their electrodense fillings and brown inclusion of tannins [ 18 ], a feature also observed in cork oak’s one-year stems [ 42 ] ( Figure 2 j).…”

“…Suberin, lignin and polyphenol production are amongst the most enriched categories during cork tissue development, but autophagy and programmed cell death (PCD) are also categories with high number of up-regulated genes [ 79 ]. Besides the high suberin content of periderm cork cells, another characteristic of these cells is the fact they are dead by the time functional maturity is reached, implying they undergo PCD [ 84 ].…”

“…Focusing on the periderm, PCD begins simultaneously as soon as fast cell wall suberization begins and phellem cells differentiate [ 18 , 88 ]. Programmed cell death occurring in cork oak has a developmental (dPCD) nature rather than being an effect of environmental factors [ 79 ].…”

Epigenetics at the crossroads of secondary growth regulation

“…The periderm formation involves several processes: phellogen initiation and cell proliferation, radial cell elongation, secondary cell wall formation through the deposition of suberin and waxes, and dPCD (Inácio et al, 2021;Serra et al, 2022). Despite the enormous progress in understanding the molecular mechanisms underlying vascular cambium regulation, very few regulators or potential regulators of phellogen activity and phellem differentiation have been identified (Miguel et al, 2016;Verdaguer et al, 2016;Xiao et al, 2020;Ye et al, 2021).…”