3.3 Ontogeny of the phloem in the stems of Austrobaileya scandens
Cross sections of stems at different axial positions revealed ontogenetic differences in the eustele, which correlated with stem mechanical properties. In the flexible searcher stems, a large pith was surrounded by discontinuous primary xylem traces consisting of only a few tracheids (Figure 5a), but a more continuous phloem layer (Figure 5b). Twining correlated with a sharp increase in the stiffness due to lignification of the pith and the development of an extra phloem pericyclic fiber layer (Figure 5c). In wider stems (the ones that traverse longest distances), the central pith constituted the largest fraction of the cross-sectional area, and the axial secondary phloem displayed a lobed morphology (Figure 5d-e), with sieve tubes closer to the vascular cambium and separated from the pericyclic fibers by parenchyma (Figure 5f).
In line with the demands for climbing and the need for flexible searcher branches, a galacturonan-rich pectic epitope, previously related to the flexibility of sieve tubes (Torode et al., 2018), localized in the sieve tube walls of the searcher, flexible stems of A. scandens (Figure 6; note that we could not perform immunolocalization in the secondary phloem due to poor preservation of the twining stems). Different cell morphologies compose the stems in cross section (Figure 6a), but signal from this epitope specifically showed the scattered distribution of sieve tubes (Figure 6b,c). Upon closer inspection, the signal of the antibody corresponded with the areas of the sieve tubes between the plasma membrane and the cell walls (Figure 6d-f). In longitudinal section, the epitope was helpful distinguishing sieve tubes from other phloem cell types (Figure 6g), which highlighted their longitudinal silhouette (Figure 6h-i), showing slightly tangential sieve plate connections (Figure 6j-l).
In the stems supported by twining, and concomitant with leaf expansion, the morphology of the sieve elements varied, gradually increasing the number of sieve plate connections at the end of the conduits (Figure 7a,b). In the thickest stems, sieve elements had extremely angled tangential connections with a great proportion of the tubes covered by sieve areas (Figure 7b,c). Numerous small pores (0.20µm on average, n=600), whose radius was similar to those of the leaf petioles, populated the sieve areas (Figure 7c,d; Behnke, 1986). In addition to morphology, sieve conduit dimensions increased from thinner to thicker stems, but their length was shorter at the base of the vine (Figure 7e).