Bordered pits in xylem of vesselless angiosperms and their possible misinterpretation as perforation plates

Abstract

Vesselless wood represents a rare phenomenon within the angiosperms, characterizing Amborellaceae, Trochodendraceae and Winteraceae. Anatomical observations of bordered pits and their pit membranes based on light, scanning and transmission electron microscopy (SEM and TEM) are required to understand functional questions surrounding vesselless angiosperms and the potential occurrence of cryptic vessels. Interconduit pit membranes in 11 vesselless species showed a similar ultrastructure as mesophytic vessel-bearing angiosperms, with a mean thickness of 245 nm (± 53, SD; n = six species). Shrunken, damaged and aspirated pit membranes, which were 52% thinner than pit membranes in fresh samples (n = four species), occurred in all dried-and-rehydrated samples, and in fresh latewood of Tetracentron sinense and Trochodendron aralioides. SEM demonstrated that shrunken pit membranes showed artificially enlarged, > 100 nm wide pores. Moreover, perfusion experiments with stem segments of Drimys winteri showed that 20 and 50 nm colloidal gold particles only passed through 2 cm long dried-and-rehydrated segments, but not through similar sized fresh ones. These results indicate that pit membrane shrinkage is irreversible and associated with a considerable increase in pore size. Moreover, our findings suggest that pit membrane damage, which may occur in planta, could explain earlier records of vessels in vesselless angiosperms.

High porosity with tiny pore constrictions and unbending pathways characterize the 3D structure of intervessel pit membranes in angiosperm xylem

“…Although it is unclear whether or not constriction size dimensions change due to an ionic effect (Lee, Holbrook, & Zwieniecki, ), the actual injection was done with the 0.1 mM PBS solution of colloidal gold. Moreover, similar experiments in which stem samples of various angiosperm species were flushed with a 10 mM KCl solution or distilled water prior to gold perfusion, did not show a major difference in the perfusion capacity of gold particles (Choat et al, ; Choat et al, ; Zhang et al, ; Table ). We prepared 1 ml of a 1:1:1:1 mixture of colloidal gold solutions, with gold particles that had an average diameter of 5 nm (± 2 nm, lot number MKCD4752), 10 nm (± 2 nm, lot number MKCC2817), 20 nm (± 2 nm, lot number MKBZ7332V) and 50 nm (± 3 nm, lot number MKCB4933).…”

“…A standard protocol was followed to prepare ultrathin sections for TEM (Jansen et al, ; Zhang et al, ). Briefly, small cubes of xylem (1 × 2 × 2 mm) from the current growth ring were cut under water, fixed overnight in a standard fixative solution (2.5% glutaraldehyde, 0.1 mol phosphate buffer, 1% sucrose and pH 7.3) in a refrigerator, and washed three to four times with phosphate buffered saline (PBS).…”

“…This view, however, requires a 3D view of pit membrane pores with multiple constrictions, which is fundamentally different from the oversimplified 2D view of pit membrane pores, with air‐seeding occurring through the single, largest pore (Jansen et al, ). Moreover, dehydration of a pit membrane can change the density and arrangement of its microfibril aggregates and may cause irreversible shrinkage (Hillabrand, Hacke, & Lieffers, ; Li et al, ; Zhang, Klepsch, & Jansen, ; Kotowska et al, 2019). Pit membranes were reported to shrink by 28% when comparing fresh samples (i.e.…”

“…However, constriction sizes examined in fresh and wet (i.e. never dried) samples of some angiosperms based on perfusion with different particles show a much smaller variation, ranging from 5 to 37 nm (Table ; Choat et al, ; Choat et al, ; Jarbeau, Ewers, & Davis, ; Williamson & Milburn, ; Zhang et al, ). Colloidal gold perfusion in fresh samples of seven angiosperm species also showed constriction sizes between 5 and 20 nm (Choat et al, ; Choat et al, ; Zhang et al, ).…”

“…This study aims to investigate the porous medium characteristics of intervessel pit membranes by applying a modelling approach that is based on comparing the thickness of fresh with completely dried and shrunken pit membranes. Earlier work on pit membranes (Jansen et al, ; Jansen et al, ; Li et al, ; Schmid & Machado, ; Zhang et al, ) allowed us to develop a shrinkage model to estimate the mean pore space between microfibril aggregates in fresh, fully hydrated pit membranes and dried, shrunken pit membranes. It is expected that porosity values of pit membranes are relatively high, similar to other fibrillar, non‐woven porous media in nature (Shou, Fan, & Ding, ).…”