FIGURES AND LEGENDS
FIGURE 1. (A) Adult vines of Austrobaileya scandens in the greenhouse. (B ) A. scandens vines at the canopy layer in the tropical rainforest of Queensland (Australia). (C ) Apical part of a ‘searcher’ branch in the greenhouse. (D ) Branches twisted around a stable support without increasing their diameter. (E ) The average leaf area at each particular node increased by five fold up to the fully expanded leaves of the seventh node (p <0.05).
FIGURE 2. Vascular anatomy of the continuum petiole-mid vein ofAustrobaileya scandens leaves. (A ) Mature leaf of A. scandens . (B ) Vascular tissues of the fiber less petiole. (C ) Vasculature in the mid part of the major vein showing a massive layer of pericyclic fibers surrounding the xylem and the phloem. (D ) Vasculature at the tip of the mid vein. (E ) Cross sectional areas of the vascular tissues at the three positions in the midvein: while the cross-sectional areas of xylem (red squares) and phloem (yellow circles) decreased linearly toward the tip (p <0.05), the fibers (grey ribbons) occupied a wide cross-sectional area in the leaf lamina, but not in the petiole.B-D . Cross sections stained with aniline blue to detect callose of the sieve tube elements. Scale bars: A = 1cm;B -D = 200 µm.
FIGURE 3. Geometrical scaling of the vascular elements in the leaves of Austrobaileya scandens . (A ) General view of the leaf veins showing the sampling areas (pink hexagons) and closeup of the minor veins. (B-C ) Length (squares) and radius (circles) of the individual vessel elements (red), and sieve tube elements (yellow) across vein orders; error bars represent standard error at ap <0.05. Inset in panel (A) shows a fluorescence image section of the leaf stained with the Feulgen reactive, cleared, and cuticle dissected. Fou, fourth order veins; pet, petiole; pri, primary vein; sec, secondary vein; ter, tertiary vein. Scale bar = 500µm.
FIGURE 4. Velocity of the phloem sap in Austrobaileya scandens leaves. Time-lapse images of a secondary vein showing a 2 min advancement of esculin hydrate dye from 0min (A ) to 6min (D ). Insets show the thresholding applied to the sequential images for the calculation of velocity, with a gradual accumulation of the dye within the veins. Scale bars = 1000μm.
FIGURE 5. Anatomy of the Austrobaileya scandens stems. (A ) Cross section of a 2mm diameter ‘searcher’ branch tip with primary growth, with an extensive central pith. (B ) Same stem showing callose in the continuous phloem ring (fluorescent green). (C ) Cross section of a stiff 2mm diameter branch showing a dramatic increase in lignification of the pith, the xylem, and a pericyclic fiber cap (fluorescent green). (D ) The phloem forms a thin layer between the xylem and the fiber cap. (E ) Wider stems (8mm diameter) with a high degree of lignification in all central tissues, wide vessels of the xylem, and enlarged areas between the xylem and the fiber cap. (F ) The fascicular phloem tissue was separated by axial multilayered phloem parenchyma and the active tubes were in the vicinity of the vascular cambium. A,C,E: cross sections of stems stained with acridine orange, which displays fluorescence of the lignified tissues in fluorescent green and living tissues in reddish color; B,D,F: cross sections of the stems stained with aniline blue for callose in the sieve tube elements of the phloem. F, fibers; p, pith; phl, phloem; xyl, xylem. Scale bars:A-F = 1mm; insets = 100µm.
FIGURE 6. Immunolocalization of LM26 epitope in the sieve tube elements of searcher branches of Austrobaileya scandens . (A ) Transverse section showing multicellular arrangement in the different tissue layers, including three isolated tracheids that compose the primary xylem (red arrowheads). (B ) Same section displaying the sieve tube elements of the primary phloem forming a continuous ring (yellow arrows). (C ) Merged images. (D ) Close up of the phloem in cross section. (E ) The profile of the sieve tubes in cross section after immunolocalization (yellow arrows). (F ) Merged images. (G ) Longitudinal section of the branch displaying he external part in the top and the internal tissues at the bottom. (H ) Longitudinal profile of the sieve tube element wall after immunolocalization, yellow arrowheads define the connection between tubes. (I) Merged images. (J) Detail of the sieve tube connections. (K) Sieve plate in the primary sieve tubes are slightly tangential (yellow arrowhead). (L) Merged images. 4µm thick transverse (A-F ) or longitudinal (G-L ) sections of the flexible branches displaying autofluorescence with the 405nm filter (A,D,G,J ), immunolocalized with the LM26 monoclonal antibody (B,E,H,K ), and merged images (C,F,I,L ). Ct, cortical tissue; phl, phloem; sp, sieve plate; vc, vascular cambium; xyl, xylem. Scale bars:A-C = 50 µm; G-I = 25 µm; D-F , J-L = 10 µm.
FIGURE 7 . Morphology of the sieve tube elements in the stems ofAustrobaileya scandens . (A ) Longitudinal view of a sieve tube element from a flexible branch, 2mm diameter, showing either simple or compound sieve plate connections (arrows). (B ) Sieve tube element of an 8mm diameter stem showing numerous sieve plate connections between tubes (arrows). (C ) Longitudinal view of a sieve tube element with scanning electron microscopy displaying numerous sieve plates and sieve areas all along the tube wall (arrows). (D ) Close ups of three sieve plates (analogous to the white dotted square in figure C), with details of pores. (E ) Sieve tube element length (squares), and width (circles) across axial stems of different diameter ranges; bars represent the standard error at a p <0.05.A, B , longitudinal hand sections of the stems stained with aniline blue to detect callose (bright fluorescence); C,D , scanning electron microscopy images showing the sieve plates and the sieve pores in detail. Scale bars: A-C = 20um; D = 2µm.
FIGURE 8. Sieve tube hydraulic resistance across the aerial organs of Austrobaileya scandens . The sieve tube resistance drops from 1.5 to 2 orders of magnitude from the minor leaf veins (squares) to the base of the stems (circles), even though the distance between them could be up to 20m. Numbers indicate the relative position along the transport pathway, noted in the figures, but they do not represent real distances.