Initial Bud Outgrowth Occurs Independent of Auxin Flow from Out of Buds

Abstract

Apical dominance is the process whereby the shoot tip inhibits the growth of axillary buds along the stem. It has been proposed that the shoot tip, which is the predominant source of the plant hormone auxin, prevents bud outgrowth by suppressing auxin canalization and export from axillary buds into the main stem. In this theory, auxin flow out of axillary buds is a prerequisite for bud outgrowth, and buds are triggered to grow by an enhanced proportional flow of auxin from the buds. A major challenge of directly testing this model is in being able to create a bud- or stem-specific change in auxin transport. Here we evaluate the relationship between specific changes in auxin efflux from axillary buds and bud outgrowth after shoot tip removal (decapitation) in the pea (Pisum sativum). The auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA) and to a lesser extent, the auxin perception inhibitor p-chlorophenoxyisobutyric acid (PCIB), effectively blocked auxin efflux from axillary buds of intact and decapitated plants without affecting auxin flow in the main stem. Gene expression analyses indicate that NPA and PCIB regulate auxin-inducible, and biosynthesis and transport genes, in axillary buds within 3 h after application. These inhibitors had no effect on initial bud outgrowth after decapitation or cytokinin (benzyladenine; BA) treatment. Inhibitory effects of PCIB and NPA on axillary bud outgrowth only became apparent from 48 h after treatment. These findings demonstrate that the initiation of decapitation- and cytokinin-induced axillary bud outgrowth is independent of auxin canalization and export from the bud.

Figure 1.

Effects of decapitation and NPA on auxin efflux from node 5 axillary buds into the main stem. A, Pea plants with seven leaves fully expanded were decapitated (decap) 10 mm above node 5 (A, right). Treatment site and region were sampled for [³H]IAA quantification. Three hours after decapitation, node 5 axillary buds were treated with 0.25 μL of [³H]IAA alone or in solution with 1 mm NPA. Two hours after treatment, internode tissue 3 to 27 mm below node 5 was sampled in 3-mm segments and quantified for radioactivity. B, Total [³H]IAA exported out of buds; different letters (a, b) on the top of the columns indicate statistically significant differences (P < 0.05; one-way ANOVA with Tukey’s correction). C, [³H]IAA exported out the buds as quantified in adjacent 3-mm stem segments. Data are means ± se (n = 3 to 6).

Figure 2.

Reduced auxin signaling (1 mm PCIB) and auxin transport (1 mm NPA) reduces auxin efflux from axillary buds on intact plants (A and B) and in decapitated plants (C and D). A and C, Total [³H]IAA recovered in the stem after 3 h; different letters (a, b) on the top of the columns indicate statistically significant differences (P < 0.05; one-way ANOVA with Tukey’s correction). Data are means ± se (n = 4). B and D, [³H]IAA exported out the buds as quantified in adjacent 3-mm stem segments.

Figure 3.

PCIB and NPA enter axillary buds and rapidly regulate auxin marker genes. Node 5 buds of L107 pea plants with seven leaves fully unfolded were treated for 3 h with 1 mm PCIB or 1 mm NPA. Expression of auxin marker genes in the bud is represented relative to the mock (100% ethanol). A and B, Auxin transport genes ABCB19 and PIN1. C and D, Auxin-inducible genes GH3 and SAUR1. E and F, Auxin biosynthesis genes YUC1 and YUC4. EF1α and GADPH were used as the internal reference genes. Data are means ± se (n = 5 pools of 4 plants).

Figure 4.

PCIB or NPA treatment to axillary buds has no effect on polar auxin transport (PATS) in the main stem. A, 0.25 μL of [³H]IAA was applied to the shoot tip of pea plants with seven leaves fully expanded and 120-mm distance between node 5 and the shoot tip. B, eleven hours after [³H]IAA application, 0.25 μL of either mock solution (100% ethanol), 1 mm NPA, or 1 mm PCIB was supplied to axillary bud on node 5. B and C, Two hours after NPA or PCIB application, internode tissue 30 mm above and below node 5 was sampled in 6-mm segments and quantified for radioactivity. B, Total [³H]IAA in the sampled stem segments; the letter “a” on the top of the columns indicates statistically significant differences (P < 0.05; one-way ANOVA with Tukey’s correction). Data are means ± se (n = 4). C, [³H]IAA in the sampled stem sections as quantified in continuous 3-mm stem segments.

Figure 5.

PCIB, NPA, and the distance at which plants were decapitated above node 5 had no effect on early bud growth after decapitation. Illustrated are the effects of 1 mm of either NPA or PCIB on the node 5 axillary bud outgrowth in pea plants. A, The position is shown at which the plants were decapitated, 1 cm (low) or 10 cm (high) above node 5. B and C, Bud growth responses are shown to 2 μL of treatments to the node 5 bud, including 1 mm PCIB (B) and 1 mm NPA (C).

Figure 6.

Effects of PCIB and NPA on initial and sustained axillary bud outgrowth. A, Pea plants with seven leaves fully expanded were decapitated 10mm above node 5 and treated with 2 μL of either a mock solution (100% ethanol), 1 mm NPA, or 1 mm PCIB. Bud lengths were measured daily over a period of 3 d after treatment. Data are means ± se (n = 12). B, Axillary buds at node 5 of pea plants with seven leaves fully expanded were treated with 2 μL of 50 μm BA alone (or in solution with 1 mm NPA). Bud lengths were measured 2 and 4 d after treatment. Data are means ± se (n = 4).