A Structurally Specialized Uniform Wall Layer is Essential for Constructing Wall Ingrowth Papillae in Transfer Cells

Abstract

Transfer cells are characterized by wall labyrinths with either a flange or reticulate architecture. A literature survey established that reticulate wall ingrowth papillae ubiquitously arise from a modified component of their wall labyrinth, termed the uniform wall layer; a structure absent from flange transfer cells. This finding sparked an investigation of the deposition characteristics and role of the uniform wall layer using a Vicia faba cotyledon culture system. On transfer of cotyledons to culture, their adaxial epidermal cells spontaneously trans-differentiate to a reticulate architecture comparable to their abaxial epidermal transfer cell counterparts formed in planta. Uniform wall layer construction commenced once adaxial epidermal cell expansion had ceased to overlay the original outer periclinal wall on its inner surface. In contrast to the dense ring-like lattice of cellulose microfibrils in the original primary wall, the uniform wall layer was characterized by a sparsely dispersed array of linear cellulose microfibrils. A re-modeled cortical microtubule array exerted no influence on uniform wall layer formation or on its cellulose microfibril organization. Surprisingly, formation of the uniform wall layer was not dependent upon depositing a cellulose scaffold. In contrast, uniform wall cellulose microfibrils were essential precursors for constructing wall ingrowth papillae. On converging to form wall ingrowth papillae, the cellulose microfibril diameters increased 3-fold. This event correlated with up-regulated differential, and transfer-cell specific, expression of VfCesA3B while transcript levels of other cellulose biosynthetic-related genes linked with primary wall construction were substantially down-regulated.

Ethylene and hydrogen peroxide regulate formation of a sterol-enriched domain essential for wall labyrinth assembly in transfer cells

“…(A) Cotyledons were cultured on MS medium containing the sterol biosynthesis inhibitor, fenpropimorph, for 3 h at 26 °C, then washed 3×5 min in dH 2 O before being transferred to MS medium in the absence/presence of the endocytosis inhibitor, dynasore, for 4 h at 4 °C and subsequently cultured for a further 3 h at 26 °C.(B) Cotyledons were cultured on MS medium for 9 h to ensure deposition of the uniform wall layer (Xia et al , 2017), before being transferred to MS medium containing fenpropimorph for 3 h at 26 °C, then washed 3×5 min in dH 2 O before being transferred to MS medium in the absence/presence of dynasore for 4 h at 4 °C and subsequently cultured for a further 3 h at 26 °C.Immediately before and following exposure to dynasore, transverse sections of treated cotyledons were stained with the membrane dye FM4-64FX. Fluorescence was measured as total pixels in specified ETC regions.…”

Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells

“…Whether H 2 O 2 action on WI papillae formation was direct or indirect was addressed as follows. Cultured cotyledons were transferred to media containing ± n -propyl gallate once UWL formation was completed at 9 h, while recruiting cells to deposit WI papillae continued for the subsequent 6 h (Xia et al , 2017). In the presence of n -propyl gallate, further WI papillae formation was arrested (Fig.…”

“…Ultrathin sections were visualized with a JEOL 1200 EX II transmission electron microscope (JEOL, Japan). Percentages of ETCs with a UWL along with estimates of original and UWL volumes per ETC were determined (see Xia et al , 2017). For SEM (Phillips, The Netherlands) to visualize WI papillae, see Zhang et al (2015 c ).…”

“…Following incubation, cotyledons were washed (4× 3 min) with 50 mM cacodylate buffer. Tissue segments were fixed for 2 h in 1.25% (v/v) glutaraldehyde and 1.25% (w/v) formaldehyde in 100 mM cacodylate buffer (pH 7.2), washed (3 ×10 min) with 100 mM cacodylate buffer, and post-fixed with 1% (w/v) osmium tetroxide in 50 mM cacodylate buffer for 1 h all at room temperature, followed by dehydration, resin embedment, and sectioning for TEM (see Xia et al , 2017). TEM micrographs of ETCs were optimized for total pixel intensity measurement by setting to a common cytoplasmic brightness (using the Expose option in Photoshop).…”

“…Given the strong transcriptional influence of apo ROS in trans -differentiating ETCs of V. faba cotyledons (Zhang et al , 2017 b ) and unanswered issues embodied in our previous study of ROS-regulated WL formation (Andriunas et al , 2012; Xia et al , 2012; see below), it was considered timely to verify the ROS responsible for regulating WL formation and identify the enzymes forming the polarized apo ROS signal and their cellular/subcellular organization. The studies by Andriunas et al (2012) and Xia et al (2012) did not exclude the possibilities that: (i) the superoxide ion radicle (O 2 – ) or the hydroxyl radicle (∙OH) regulate deposition of the WL (Kärkönen and Kuchitsu, 2015); (ii) at a concentration of 100 μM, diphenyleneiodonium chloride (DPI) could well have inhibited ROS biosynthetic enzymes other than respiratory burst oxidase homologues (rbohs), such as class III peroxidases (Prxs), copper amine oxidases (CAOs), and polyamine oxidases (PAOs; Kärkönen and Kuchitsu, 2015); and (iii) whether ROS action on WI papillae formation was direct or indirect resulting from regulating UWL deposition which forms an essential platform for WI papillae construction (Xia et al , 2017). In addition to addressing these questions, we made use of the ETC transcriptome to identify gene candidates expressing H 2 O 2 biosynthetic and catabolic enzymes (Zhang et al , 2017 b ).…”