Auxin flow-mediated competition between axillary buds to restore apical dominance

Abstract

Apical dominance is one of the fundamental developmental phenomena in plant biology, which determines the overall architecture of aerial plant parts. Here we show apex decapitation activated competition for dominance in adjacent upper and lower axillary buds. A two-nodal-bud pea (Pisum sativum L.) was used as a model system to monitor and assess auxin flow, auxin transport channels, and dormancy and initiation status of axillary buds. Auxin flow was manipulated by lateral stem wounds or chemically by auxin efflux inhibitors 2,3,5-triiodobenzoic acid (TIBA), 1-N-naphtylphtalamic acid (NPA), or protein synthesis inhibitor cycloheximide (CHX) treatments, which served to interfere with axillary bud competition. Redirecting auxin flow to different points influenced which bud formed the outgrowing and dominant shoot. The obtained results proved that competition between upper and lower axillary buds as secondary auxin sources is based on the same auxin canalization principle that operates between the shoot apex and axillary bud.

Figure 1. Axillary buds released from dormancy compete for dominance in pea.

(a) Scheme of intact plant. Red arrows represent auxin (IAA) flow; red arrows crossed with black X represent disabled auxin flow. Auxin loaded from the apex (as primary source) to the stem prevents auxin canalization and its export from the axillary buds (as potential secondary auxin sources). (b) Scheme of decapitated plant. Red and crossed red arrows as depicted in a). Dashed crossed red arrow represents intermitted auxin flow after temporary activation. Green arrow represents bud outgrowth and dominance, dashed green arrow represents temporary outgrowth. Apex, the primary source for auxin flow, is removed and auxin synthesized in the buds can be exported, resulting in outgrowth of both buds. The initial outgrowth turns into competition leading to upper bud dominance over the lower. (c) Intact control plant 7-DAS (at the beginning of experiment). (d) Plant 5 days after decapitation with outgrowing and dominant upper axillary bud and temporarily outgrown and then arrested lower bud. (e) Intact plant of same age (7-DAS + 5 days); both axillary buds remain arrested. (f) Length of axillary buds and forming shoots, where: (li) lower bud of intact plants; (ui) upper bud of intact plants; (ld) lower bud of decapitated plants; (ud) upper bud of decapitated plants. Statistically significant differences (identified by Student’s t-test): α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 60). (g) Relative expression of PsDRM1 gene in lower and upper axillary buds following decapitation. Statistically significant differences (identified by Student’s t-test): α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 4). (h,j) Immunoanalysis of PIN1 auxin efflux carriers (red signal) showed polar localization in the primary stem (h), lack of localization in procambial cells of inhibited axillary buds, (i) and polar localization in procambial cells of outgrowing buds (j). Scale bar, 100 μm.

Figure 2. Auxin pool in decapitated stem delays release of buds from dormancy.

(a,b) Scheme of plant with long stump decapitated 90 mm above the upper bud (a) and plant with short stump decapitated 5 mm above the upper bud (b). Red arrows represent auxin (IAA) flow; red arrows crossed with black X represent disabled auxin flow. Green arrows represent bud outgrowth. Auxin depletion or decrease from the missing apex has impact on bud outgrowth timing. (c,d) Relative expression of PsDRM1 (c) and PsBRC1 (d) genes in the upper axillary bud of plants with long or short stump. Statistically significant differences (identified by Student’s t-test): α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 4).

Figure 3. PAT Interruption in the primary stem releases buds from dormancy.

(a) Scheme of plant wounded above the upper bud. Red arrows represent auxin (IAA) flow; red arrow crossed with black X represents disabled auxin flow. Green arrow represents bud outgrowth. Weakening stem auxin flow facilitated auxin export from the upper bud and its outgrowth. The lower bud remained arrested in dormancy by auxin loaded from the upper bud. (b) Scheme of intact plant subapically treated with TIBA-ring. Arrows as depicted in a). Auxin efflux inhibitor blocks auxin flow from the apex and the upper bud becomes a new auxin source, which continues to prevent auxin transport from the lower bud. (c) Wounded plant. Black arrow points to the lateral shoot formed from the upper axillary bud, above which the stem was incised. (d) Length of axillary buds and forming shoots, where: (li) lower bud of intact plants, (ui) same plants, upper bud, (liTs) lower bud of intact plants subapically treated with TIBA-ring, (uiTs) same treatment, upper bud. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 60). (e) Relative expression of PsDRM1 gene in the lower and upper axillary bud of intact plants subapically treated with TIBA-ring. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 4). (f) [³H]-IAA transport from the apex in stem subapically treated with TIBA-ring was measured in two stem sections at a distance of 0–4 and 4–8 mm under the TIBA application site. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 10). (g) PIN1 auxin efflux carrier immunoanalysis (red signal) in stem cells at TIBA-ring position exhibited no visible changes in organization. Stage 24 h after treatment. Scale bar, 100 μm.

Figure 4. Interruption of PAT between buds releases lower bud from dormancy.

(a) Scheme of plant wounded above the lower bud. Red arrows represent auxin (IAA) flow; red arrow crossed with black X represents disabled auxin flow. Green arrow represents bud outgrowth. Weakening stem auxin flow facilitated auxin export from the lower bud and its outgrowth. (b) Scheme of intact plant treated with TIBA-ring between the buds. Arrows as depicted in a). Auxin efflux inhibitor prevents auxin flow in the stem and allows auxin export from the lower bud and its outgrowth. (c) Scheme of decapitated plant treated with TIBA-ring between the buds. Arrows as depicted in a). Competition for outgrowth between buds is relinquished by TIBA, which isolates the lower bud from the auxin loaded by the upper bud, resulting in two equally growing shoots. (d) Wounded plant. Black arrow points to the lateral shoot was formed from the lower bud, above which the stem was incised. (e) Length of axillary buds and forming shoots, where: (li) lower bud of intact plants, (ui) same plants, upper bud, (liTb) lower bud of intact plants treated with TIBA-ring between the buds, (uiTb) same treatment, upper bud. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 60). (f) Length of axillary buds and forming shoots, where: (ld) lower bud of decapitated plants, (ud) same plants, upper bud, (ldTb) lower bud of decapitated plants treated with TIBA-ring between the buds, (udTb) same treatment, upper bud. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 60). (g) Relative expression of PsDRM1 gene in lower and upper buds of decapitated plants treated with TIBA-ring between the buds. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 4). (h) [³H]-IAA transport in decapitated stems from shoots formed from upper axillary buds measured in two stem sections at distance of 0–4 and 4–8 mm under the TIBA-ring between the buds. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 10).

Figure 5. Inhibition of PAT from the upper bud releases lower bud from dormancy.

(a) Scheme of decapitated plant treated with TIBA-, NPA-, or CHX-ring on the upper bud (asterisk indicates NPA or CHX was also applied). Red arrows represent auxin (IAA) flow; red arrows crossed with black X represent disabled auxin flow. Green arrow represents bud outgrowth. Treated bud outgrowth was inhibited by a mechanism associated with each applied chemical, which resulted in the lower bud becoming a new auxin source and developing a shoot. (b) Length of axillary buds and forming shoots, where: (ltiba) lower bud of decapitated plants, when upper bud was treated with TIBA, (utiba) same treatment, upper bud, (lnpa) lower bud of decapitated plants, when upper bud was treated with NPA, (unpa) same treatment, upper bud, (lchx) lower bud of decapitated plants, when upper bud was treated with CHX, (uchx) same treatment, upper bud. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 60). (c–e) Relative expression of PsDRM1 gene in lower and upper axillary buds following TIBA (c), NPA (d), and CHX (e) treatments in upper buds of decapitated plants. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 4). (f) [³H]-IAA export from upper axillary buds, measured in two stem sections, from 0–4 and 4–8 mm under the upper bud. Decapitation facilitated auxin export, while TIBA, NPA and CHX reduced auxin flow. Statistically significant differences (identified by Student’s t-test) α = 0.05* and α = 0.01**. Error bars represent standard deviations (n = 10). (g–i) PIN1 auxin efflux carrier immunoanalysis (red signal) in axillary buds showed TIBA (g), NPA (h), and CHX (i) lanolin paste treatments did not prevent polarization following decapitation. Stages 24 h after treatment. Scale bar, 100 μm.