Ectopic expression of LONELY GUY7 in epidermis of internodal segments for de novo shoot regeneration without phytohormone treatment in ipecac

Abstract

In many plant species, the application of exogenous phytohormones is crucial for initiating de novo shoot regeneration. However, ipecac [Carapichea ipecacuanha (Brot) L. Andersson] has a unique ability to develop adventitious shoots on the epidermis of internodal segments without phytohormone treatment. This characteristic allows us to evaluate the effects of endogenous phytohormones in this species. Here, we showed that the presence of the pith, including vascular bundles in the internodal segment, is required to activate both endogenous cytokinin (CK) biosynthesis and adventitious shoot formation. Adventitious shoots were mainly formed in the apical region of internodal segments, where the CK biosynthesis genes ISOPENTENYL TRANSFERASE 3 (CiIPT3) and LONELY GUY 7 (CiLOG7) were spontaneously upregulated in the early culture stage on phytohormone-free medium. In addition, CiIPT3 and CiLOG7 were respectively expressed in the pith and the epidermis of the internodal segments. The expression of CiLOG7 was localized as several spots on the epidermis. These findings suggest that CK precursors are generated in the pith, transferred to the epidermis, and then converted into active CKs, facilitating adventitious shoot formation on the epidermis. Conversely, auxin levels rapidly decreased during culture and remained low in the region of shoot formation. Auxin is transferred to the basal region of internodal segments, and strongly suppressed the CiLOG7 expression and decreased the CK levels. Thus, we conclude that the ectopic expression of CiLOG7 in the epidermis of internodal segments contributes to de novo shoot regeneration in ipecac.

FIGURE 1

Effects of the pith on adventitious shoot formation in ipecac. (A) Division of internodal segments into sections with or without the pith including vascular bundles. (B) Representative images of tissues after 4 weeks of culture under the indicated conditions. White arrowheads, adventitious shoots; dotted lines, areas where epidermis was excised off. Number of internodal segments with adventitious shoots formed is shown in each image. The experiment was conducted twice, with six segments per experiment. 2iP, N ⁶‐isopentenyladenine. tZ, trans‐zeatin. Bar, 2 mm.

FIGURE 2

CK biosynthesis in internodal segments with or without the pith. Tissues were collected before culture (0 day) or after 7 days of culture on a phytohormone‐free culture medium. Internodal segments (4 with and 14 without the pith) were used in each experiment. (A) Relative expression of CK biosynthesis genes. EF‐1 was used as an internal standard. The data were normalized to the expression in the internodal segment without a vascular bundle before culture. (B) Endogenous CK levels in internodal segments. 2iP, N ⁶‐isopentenyladenine. tZ, trans‐zeatin. iPR, isopentenyladenine riboside. tZR, trans‐zeatin riboside. Data are mean ± SE (n = 4). Different letters indicate significant differences among segments (Tukey's HSD, p < 0.05; CiIPT3, CiLOG7, 2iP, iPR, tZR) or between internodal segments with versus without the pith (Student's t‐test; tZ, all p > 0.05); n.d., not detected, n.s., not significant.

FIGURE 3

Time course analysis of CK biosynthesis in internodal segments during the initial 7 days of culture. (A) Representative images of tissues after 0 day and 35 days of culture on hormone‐free culture medium. White arrowheads, adventitious shoots. Bar, 2 mm. (B) Relative expression of CK biosynthesis genes. EF‐1 was used as an internal standard. The data were normalized to the CiIPT3 expression in the apical region before culture. Data are mean ± SE (n = 4). Internodal segments were collected before culture (0 day), after 1 h, 3 h, 6 h, 12 h, 1 day, 2 days, 3 days, 5 days, and 7 days of culture on phytohormone‐free culture medium. Then, the internodal segments were divided into the apical region and basal region sections. Five segments were used in each experiment. AR: apical region, BR: means basal region. (C) Endogenous CK levels in internodal segments. Insets show enlarged 0–6‐h plots. Four segments were used in each experiment. Data are mean ± SE (n = 3). Asterisk indicates significant difference compared with apical region (t‐test, p < 0.05).

FIGURE 4

Relative expression of CK biosynthesis genes in the pith, cortex, and epidermis of internodal segments. (A) Cryo‐sections after laser microdissection. Red dotted lines indicate the area excised by laser microdissection. Bar, 500 μm. (B) Relative expression of IPT3 and LOG7 in each section. Five segments were used in each experiment. Data are mean ± SE (n = 4). EF‐1 was used as an internal standard. Different letters indicate significant differences (Tukey's HSD, p < 0.05).

FIGURE 5

In situ hybridization analysis of CiLOG7 on cross‐sections of internodal segments. The segments were cultured on a phytohormone‐free culture medium for 3 days or 7 days. The apical region (region I) was used. Arrowheads, LOG7 expression in the epidermis after 7 days of culture. Bars, 200 μm.

FIGURE 6

Effects of NAA treatment on CK biosynthesis in internodal segments. (A) Representative images of tissues after 35 days of culture under the indicated conditions. White arrowheads, adventitious shoots. Bar, 2 mm. (B) Relative expression of CK biosynthesis genes. EF‐1 was used as an internal standard. Tissues were collected after 3 days of culture on a culture medium with or without 50 nM NAA. Then, the internodal segments were divided into the apical region and basal region sections. Five internodal segments were used in each experiment. (C) Endogenous CK levels in internodal segments. Data are mean ± SE (n = 3). Different letters indicate significant differences among segments (Tukey's HSD, p < 0.05, tZ, tZR, iPR). Asterisk indicates a significant difference compared with apical region (t test, p < 0.05). n.d.: not detected, n.s.: not significant.