Spatial Distribution of Selected Chemical Cell Wall Components in the Embryogenic Callus of Brachypodium distachyon

Abstract

Brachypodium distachyon L. Beauv. (Brachypodium) is a species that has become an excellent model system for gaining a better understanding of various areas of grass biology and improving plant breeding. Although there are some studies of an in vitro Brachypodium culture including somatic embryogenesis, detailed knowledge of the composition of the main cell wall components in the embryogenic callus in this species is missing. Therefore, using the immunocytochemical approach, we targeted 17 different antigens of which five were against the arabinogalactan proteins (AGP), three were against extensins, six recognised pectic epitopes and two recognised hemicelluloses. These studies were complemented by histological and scanning electron microscopy (SEM) analyses. We revealed that the characteristic cell wall components of Brachypodium embryogenic calli are AGP epitopes that are recognised by the JIM16 and LM2 antibodies, an extensin epitope that is recognised by the JIM11 antibody and a pectic epitopes that is recognised by the LM6 antibody. Furthermore, we demonstrated that AGPs and pectins are the components of the extracellular matrix network in Brachypodium embryogenic culture. Additionally, SEM analysis demonstrated the presence of an extracellular matrix on the surface of the calli cells. In conclusion, the chemical compositions of the cell walls and ECMSN of Brachypodium callus show spatial differences that correlate with the embryogenic character of the cells. Thus, the distribution of pectins, AGPs and hemicelluloses can be used as molecular markers of embryogenic cells. The presented data extends the knowledge about the chemical composition of the embryogenic callus cells of Brachypodium.

Extracellular matrix and wall composition are diverse in the organogenic and non-organogenic calli of Actinidia arguta

“…In addition, during the somatic embryogenesis of Trifolium nigrescens Viv., it was demonstrated that the low methylesterified homogalacturonan (HG), which is recognised by the JIM5 antibody, and the arabinan from side chains of rhamnogalacturonan I (RG I), which is recognised by the LM6 antibody, were detected in the embryogenic sectors of the explant (Pilarska et al 2013). Moreover, the chemical composition of the cell walls and ECM of a Brachypodium callus displayed spatial differences that correlated with the embryogenic character of the cells (dense cytoplasm, high nucleus/cytoplasm ratio, large nucleoli), thus indicating that the distribution of pectins, AGPs and hemicelluloses can be used as molecular markers of the embryogenic cells (Betekhtin et al 2016).…”

“…It has been postulated that the residues of galacturonic acid that are present in the chains of low or unmethyl-esterified HG are cross-linked with calcium cations, thus forming a "pectin gel", which, in turn, may contribute to wall stiffening (Willats et al 2001a;Jiang et al 2005;Caffall and Mohnen 2009;Hongo et al 2012). The presence of the above-mentioned pectic epitopes in the ECM has been observed not only on the surface of the somatic embryos and embryogenic callus but also on the OC in different plant species (Konieczny et al 2007;Popielarska-Konieczna et al 2008;Betekhtin et al 2016). Chapman et al (2000) stated that the non-esterified pectins that are present in the extracellular matrices may be responsible for maintaining embryonic cell adhesion and that the occurrence of the layer that covers the cell itself could spatially limit cell division.…”

“…Open arrows: a, i an epitope that was detected in the cytoplasmic compartments. Scale bars: a-h = 10 µm, i = 50 µm ◂ such an extracellular localisation of the extensins is not unique-extensins were detected on the surface of Brachypodium callus cells (Betekhtin et al 2016) and in an Arabidopsis graft union (Sala et al 2019).…”

In Vitro Tissue Culture in Brachypodium: Applications and Challenges

“…Embryogenic cells have the typical features of undifferentiated cells such as a small or poorly developed vacuole, dense cytoplasm and a nucleus that usually has one and occasionally two nucleoli [98]. Mitoses are also occasionally present [99]. In contrast, non-embryogenic calli have parenchymatous or differentiated cells that are larger and highly vacuolated.…”

“…An embryogenic callus that was formed by cells that had been derived from the protodermal-dividing cells of the scutellum [98] initially had typical meristematic features and a high level of protein accumulation, which is connected with the acquisition of a competent state [100]. Embryogenic calli have an extracellular matrix on the surface of the calli cells (ECMSN), which is composed of the arabinogalactan proteins (AGPs) and pectins [99]. The pectins, AGPs and hemicelluloses can be used as molecular markers of the embryogenic cells.…”