Figure 1.
Radiolabeled auxin transport assays.
Radiolabeled auxin (3H-IAA) or benzoic acid (3H-BA) were applied to Populus stems (100 nM in 1.5% agar) in a series of transport assays. Inner and outer compartments were created with thin metal rings to separate primary and secondary vascular tissues, respectively. Four separate assays were performed in order to test (a) the capacity of primary versus secondary tissue for basipetal auxin transport, (b) the connection between leaf traces and the two respective compartments, (c) the potential for exchange between primary and secondary tissues, and (d) the potential for outward radial transport of auxin. BA served as a diffusion and non-polar transport control; addition of NPA (10 µM) to the media provided further evidence of polar transport.
Figure 2.
GUS expression in PtaDR5 tissue indicates an auxin response.
GUS expression in a representative PtaDR5 line (a, b, c; CONTROL showing endogenous expression) and following incubation in 30 µM IAA (d, e, f; TREATED showing response to exogenous IAA). Stem segments (a−e) and roots (c and f) from plants grown in vitro were incubated in 1/2 strength MS growth media with or without IAA for 12 hours in the dark prior to histochemical staining with X-Gluc. The strong response to exogenous auxin (d, e, f) and pattern of endogenous GUS expression in axillary buds (a), cambial zone and primary xylem parenchyma (b; arrowhead and arrows, respectively), and primary and lateral root tips (c and inset, respectively) were consistent with other independently transformed PtaDR5 lines.
Figure 3.
Endogenous auxin response in PtaDR5 lines and IAA quantification suggest two routes for basipetal transport.
Ringing stems with NPA (50 mM in lanolin) caused an increase in signal above (a, left) the site of application; signal below the site of application (a, right) was comparable to control (inset; above mock-treatment shown on left, below on right). GUS expression in both the cambial zone and primary xylem parenchyma (PXP) continued throughout active growth, with the latter remaining up to 100 nodes beneath the apex, or as long as leaves remained firmly attached to the stem (b; stems 90 and 20[inset] nodes beneath the apex). Dormant stems lacking leaves showed no GUS expression (c). Concentrations of free IAA (ng IAA g−1 fresh tissue) in mature PXP and developing secondary xylem as quantified by GCMS support a role of PXP in IAA transport and/or signaling (d). In contrast, no free IAA was reliably detected in mature secondary xylem (i.e., the region between developing xylem and primary xylem; data not shown). Scale bars represent 1 mm.
Figure 4.
Basipetal movement of 3H-IAA and 3H-BA through Populus stem segments at two vertical positions.
Internode 35 (i.e., stem segments approximately 35 nodes beneath the shoot apex) was mid-crown with leaves that had reached full expansion. Internode 90 was near the base of the stem with small leaves that remained firmly attached. Radiolabeled compounds were supplied in agar (1.5%. at 100 nM concentrations) to the entire apical end and collected from inner and outer compartments at the basal end. The polar transport inhibitor NPA (10 µM) reduced basipetal movement of 3H-IAA through the outer compartment at both stem positions, and through the inner compartment around 35, but had no effect on movement through the inner compartment around internode 90 (n = 5 trees, with 2−3 technical replicates per tree per treatment). Refer to Figure 3b for GUS expression at comparable stages of development.
Figure 5.
Movement of 3H-IAA and 3H-BA from a cut petiole.
3H-IAA and 3H-BA were applied to cut petioles and transported to the inner and outer vascular compartments of the apical and basal ends. 3H-IAA traveled significantly faster than 3H-BA, suggesting that its movement was facilitated. The PAT inhibitor NPA had little to no effect on apical movement, whereas basipetal movement was significantly reduced, most notably in the outer compartment (n = 12 [BA and IAA] or 6 trees [IAA + NPA], with 3 technical replicates per tree per treatment collected between the 40th and 60th internode from the apex).
Figure 6.
Basipetal movement of 3H-IAA and 3H-BA within and between inner and outer vascular compartments.
Radiolabeled compounds were supplied in agar to one of the two compartments at the apical end; the other compartment received a control agar. Recovery of radiolabel from the basal end in the non-delivery compartment suggests a radial route of exchange. The polar transport inhibitor NPA reduced basipetal movement of 3H-IAA applied to and recovered from the outer compartment, but had little to no effect on recovery from the non-supply compartment (n = 3 trees, with 3 technical replicates per tree per treatment collected between the 60th and 80th internode from the apex).
Table 1.
Radial transport of IAA ± two polar auxin transport inhibitors compared to a diffusion control.
Figure 7.
GUS expression in PtaDR5 lines during the transition from primary to secondary growth.
The shoot apex was defined as the tight cluster of developing leaves above the first internode that could be clearly identified with the naked eye. This internode was defined as the first internode beneath the apex and was subtended by the first node/leaf beneath the apex. Subsequent nodes/internodes were numbered accordingly; see text for details. GUS expression in the stem at the shoot apex (a, b) was restricted to PXP (arrowheads) associated with mature and developing protoxylem and the opposing strips of procambium (bounded by arrows). The first evidence of GUS expression tangentially linking isolated poles of primary xylem (c) occurred in the third internode beneath the apex (d). Radially organized files of procambium were first clearly seen in the fifth internode beneath the apex opposite the most developed PXP poles (e), but regions between these poles in the same internode lacked radial organization; here GUS expression was found in undifferentiated cells linking the earliest developing protoxylem (f). A continuous cambium with clear radial files producing both secondary xylem and phloem was well-established in the seventh internode beneath the apex (g, h). Here the development of secondary xylem has separated regions of GUS expression in PXP and the cambial zone (g). Scale bar represents 50 µm.
Figure 8.
Vascular connections between developing leaf primordia and primary vascular bundles in the stem below are a function of phyllotaxis.
Current molecular work suggests that auxin is first transported up through the epidermis and then channeled (i.e., canalized) down through the center of emerging leaf primordia (1), and that this process precedes the differentiation of procambium (2). Classical development work has shown repeatedly that procambial differentiation procedes acropetally into the expanding primordium, continuous with the procambial strands below (*), although when and how this process initiates is not known. Similarly, classical work has shown that primary phloem is the first vascular tissue to differentiate from procambium (3), and that its development is acropetal and continuous with the primary phloem below (*). In contrast, primary xylem differentiation is discontinuous (4), lags behind primary phloem, and proceeds both acropetally and basipetally (arrowheads). The work described here suggests that strands of parenchyma (†) form a second basipetal route for PAT. Although the developmental role for this tissue is not known, it may serve as a guide for developing primary xylem, which appears bounded on both abaxial and adaxial sides by tissue involved in PAT (procambium and PXP, respectively).