Accumulation and cross-linkage of β-1,3/1,6-glucan lead to loss of basal stipe cell wall extensibility in mushroom Coprinopsis cinerea

Highlights

• Endo-β-1,3-glucanase (ENG) could restore mature basal stipe wall extensibility.

• ENG-pretreated basal stipe walls could be induced to extend by chitinase.

• ENG released more glucose from the basal stipe walls than the apical stipe walls.

• Products released from basal stipe by ENG were consistent with that of β-1,3/1,6-glucan.

• β-1,3/1,6-Glucan more accumulated and cross-linked in basal stipe than apical stipe

Abstract

The mature basal stipe of mushroom Coprinopsis cinerea loses wall extensibility. We found that an endo-β-1,3-glucanase ENG from C. cinerea could restore mature basal stipe wall extensibility via pretreatment such that the ENG-pretreated basal stipe walls could be induced to extend by chitinase ChiIII. ENG pretreatment released glucose, laminaribiose, and 3-O-D-gentiobiose-D-glucose from the basal stipe walls, consistent with ENG-digested products of β-1,6-branched β-1,3-glucan. Different effects of endo-β-1,3-glucanase ENG and exo-β-1,3-glucanase EXG pretreatment on the structure, amount and ratio (β-1,3-glucoside bonds to β-1,6-glucoside bonds) of products from the basal stipe and the apical stipe cell walls, respectively, and on the cell wall extensibility and the cell wall ultra-architecture of the basal stipes were analyzed. All results demonstrate that the more accumulation and cross-linkage of β-1,6-branched β-1,3-glucan with wall maturation lead to loss of wall extensibility of the basal stipe regions compared to the apical stipe cell walls.

Introduction

Stipe elongation growth is one of the distinctive characteristics of the growth and development of mushroom fruiting bodies. Coprinopsis cinerea is a good model mushroom for study of stipe wall extension (Liu, Bi et al., 2020; Liu, Wang et al., 2020). Stipe elongation growth mainly results from the elongation of cell length rather than an increase in cell numbers in C. cinerea (Gooday, 1985; Kamada & Takemaru, 1977; Shioya et al., 2013). When stipe cells elongate, the whole cell surface elongates (Craig, Gull, & Wood, 1977; Kües, 2000; Voisey, 2010). The stipe cell is enclosed within a cell wall, and the cell wall needs to be loosened so it can expand for stipe cell elongation. The fungal cell wall mainly consists of chitin, β-1,6-branched β-1,3-glucan, and some other polysaccharides (such as β-1,6-glucan, β-1,4-glucan, α-1,3-glucan, and mannan), as well as a variety of glycoproteins (Cabib & Arroyo, 2013; Fontaine et al., 2000; Free, 2013; Gow, Latge, & Munro, 2017; Kang et al., 2018; Latge, 2007; Patel & Free, 2019; Ruiz-Herrera, 2012). Usually, β-1,6-branched β-1,3-glucan and chitin, and even α-1,3-glucan, were suggested to form a scaffold structure in fungal cell walls. In the scaffold structure chitin molecules associate with each other through intermolecular hydrogen bonds to form chitin microfibrils that are surrounded by a matrix of β-glucans including β-1,3-glucan, β-1,6-glucan and β-1,4-glucan which were proposed to covalently or non-covalently interact with chitin or α-1,3-glucan, or each other (Cabib & Arroyo, 2013; Fontaine et al., 2000; Free, 2013; Gow et al., 2017; Kang et al., 2018; Latge, 2007; Patel & Free, 2019; Ruiz-Herrera, 2012).

Stipe elongation growth shows an apical-high gradient along the stipe from the apical region to the basal region; i.e., the apical region elongates most, the middle region shows slow elongation, and the basal region and basal swollen plectenchyma region essentially do not elongate in the stipe of C. cinerea fruiting bodies (Eilers, 1974; Niu et al., 2015; Zhang et al., 2014). Consistent with the regional dependence of elongating growth in the stipe, stipe wall extensibility shows a similar gradient distribution in the stipe; i.e., the maximal acid-induced native cell wall extension appears in the apical stipe region, while the basal and basal swollen plectenchyma regions of the stipe only show minor acid-induced native cell wall extension (Niu et al., 2015; Zhang et al., 2014).

Recently, we found that C. cinerea chitinases ChiIII and ChiE1 could reconstitute heat-inactivated apical stipe wall extension in a stable and continuous extension profile, which suggested that chitinases play a key role in stipe cell wall elongation in the C. cinerea fruiting bodies (Zhou et al., 2019). Later, we further reported that β-1,3-glucanases could cooperate with chitinases at a low concentration to reconstitute heat-inactivated apical stipe wall extension (Kang, Zhou et al., 2019, 2020). In the elongating stipe cell walls, parallel chitin microfibrils are linked to β-1,6-branched β-1,3-glucans or other polysaccharide molecules (Kang et al., 2020; Liu, Bi et al., 2020; Liu, Wang et al., 2020), and the cleavage of the cross-linking by chitinases results in the separation of the chitin microfibrils to increase the wall space for inserting newly synthesized β-1,3-glucans and chitins under turgor pressure. Remarkably, chitinases show maximal reconstituted wall extension activities in the heat-inactivated elongating apical stipe region but no or poor reconstituted wall extension activity in the heat-inactivated nongrowing basal stipe regions (Zhou et al., 2019). It is reasonable to propose that the loss of mature basal stipe cell wall extensibility is not due to the lack of hydrolyases in the mature stipe cell wall but instead is a result of the biochemical modification of the mature basal stipe wall, which loses its susceptibility to chitinases. However, little information is available regarding the differences in the structure and composition of cell walls between the growing apical stipe region and the nongrowing basal stipe region. The aim of this study is to explore what components are added to or what cross-linking bonds are formed in the cell walls that cause basal stipe cell wall maturation to lose its extensibility.