Pectin De-methylesterification and AGP Increase Promote Cell Wall Remodeling and Are Required During Somatic Embryogenesis of Quercus suber

Abstract

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement

“…As embryogenesis proceeds, decreasing methylesterification levels have been reported in cell walls of differentiating embryos, in agreement with observations in other developmental pathways from proliferating to differentiating tissues of various plant species (Jolie et al, 2010). Changes in pectin esterification levels correlate with temporal expression patterns of PME and PMEI genes in microspore embryogenesis of B. napus (Solis et al, 2016), and unpublished results) and somatic embryogenesis of Q. suber (Pérez-Pérez et al 2018b). For example, differentiating embryos (like torpedo and cotyledonary embryos) show cell walls rich in methylesterified pectins, together with high expression levels of PME (Fig.…”

“…Moreover, inhibition of PME activity by catechin (Lewis et al, 2008) impairs somatic embryogenesis in Q. suber (Pérez-Pérez et al 2018b), indicating that pectin esterification and cell wall configuration play a relevant role in embryogenesis induction and/or progression (Solis et al, 2016), Pérez-Pérez et al 2018b). Taken together, these findings support the idea that PME-mediated configuration of pectins could be a crucial factor for microspore embryo differentiation, acting via the promotion of cell wall remodelling during the process.…”

Immunoprofiling of Cell Wall Carbohydrate Modifications During Flooding-Induced Aerenchyma Formation in Fabaceae Roots

“…As a result, negatively charged carboxyl groups are created that participate in cross-linking reactions with calcium cations (Supplemental Figure 1). These crosslinking interactions form an "egg box" structure of paired homogalacturonan chains that allows susceptibility to hydrolytic enzymatic degradation of the pectin backbone from polygalacturonase (Supplemental Figure 2) and pectate lyase activity that destabilizes the cell wall matrix (Ochoa-Villarreal et al, 2012;Pérez-Pérez et al, 2019). DME activity has been previously identified during cortical aerenchyma development in several crop species such as Zea mays (maize) (Gunawardena et al, 2001a), Oryza sativa (rice) (Qu et al, 2016) and Saccharum sp.…”

Vegetative propagation of elite Eucalyptus clones as food source for honeybees (Apis mellifera); adventitious roots versus callus formation

“…Pectin and pectin methylesterification are regulated at various levels (Peaucelle et al 2015;Wolf et al 2012) and can affect cell-wall rigidity, cell expansion, and organ development in different ways (Peaucelle et al 2012;Peaucelle et al 2011;Voxeur and Hofte 2016). Differential pectin methylesterification has been shown in callus from cork oak (Quercus suber) formed in tissue culture (Pérez-Pérez et al 2019). The proembryogenic cells were rich in methylesterified pectin, while the differentiated cells of the embryo were rich in de-methylesterified pectin (Pérez-Pérez et al 2019).…”