Root cap-dependent gravitropic U-turn of maize root requires light-induced auxin biosynthesis via the YUC pathway in the root apex

Abstract

Gravitropism refers to the growth or movement of plants that is influenced by gravity. Roots exhibit positive gravitropism, and the root cap is thought to be the gravity-sensing site. In some plants, the root cap requires light irradiation for positive gravitropic responses. However, the mechanisms regulating this phenomenon are unknown. We herein report that maize roots exposed to white light continuously for ≥1-2h show increased indole-3-acetic acid (IAA) levels in the root tips, especially in the transition zone (1-3mm from the tip). Treatment with IAA biosynthesis inhibitors yucasin and l-kynurenine prevented any increases in IAA content and root curvature under light conditions. Analyses of the incorporation of a stable isotope label from tryptophan into IAA revealed that some of the IAA in roots was synthesized in the root apex. Furthermore, Zmvt2 and Zmyuc gene transcripts were detected in the root apex. One of the Zmyuc genes (ZM2G141383) was up-regulated by light irradiation in the 0-1mm tip region. Our findings suggest that IAA accumulation in the transition zone is due to light-induced activation of Zmyuc gene expression in the 0-1mm root apex region. Light-induced changes in IAA levels and distributions mediate the maize root gravitropic U-turn.

Keywords: Indole-3-acetic acid (IAA) biosynthesis; Zmvt2; Zmyuc.; light dependent; maize; root cap; root gravitropism; yucasin.

Fig. 1.

Light-dependent gravitropic response of maize roots and the necessity of the root cap. (A) Etiolated maize seedlings (2 d old) were placed horizontally on a wet floral foam block, and incubated for 20h in darkness or under white light. Left, with intact roots; right, with de-capped roots. (B) Roots of 2-day-old maize seedlings were inserted into capillary tubes and inverted, then kept for 20h in darkness or under white light. Roots were photographed after they were removed from capillary tubes. Top, 20h incubation in the dark; bottom, 20h incubation under white light. Left, intact roots; right, de-capped roots. (This figure is available in colour at JXB online.)

Fig. 2.

Effects of the duration of white light exposure on root U-turn behaviour. Roots of 2-day-old maize seedlings were inserted into capillary tubes, inverted under dim green light, exposed to white light for specific periods, and then incubated in darkness. (This figure is available in colour at JXB online.)

Fig. 3.

Light irradiation increased IAA levels in root tips. Etiolated maize seedlings (2 d old) were incubated under white light (for 1.5h; white column) or in darkness (black column). (A) Abundance of IAA in 0–1, 0–3, and 0–5mm maize root tip regions [per tip (left) and per mg fresh weight (right)]. Values are presented as the mean ±SE (0–1mm, n=3; 0–3mm, n=8; 0–5mm, n=3). Asterisks indicate significant differences between roots treated in darkness and light-irradiated roots (Student’s t-test; ***P<0.001; *P<0.05). (B) Effect of de-capitation on IAA levels in the 0–3mm root tip region. Data were obtained from two independent experiments. Values are presented as the mean ±SE (4≤n≤5). Asterisks indicate significant differences between roots treated in darkness and light-irradiated roots (**P< 0.01). (C) Photo of the maize root apex and schematic views of root structure. EZ, elongation zone; TZ, transition zone; MS, meristem; RC, root cap. (This figure is available in colour at JXB online.)

Fig. 4.

Effects of several IAA-related inhibitors on IAA levels in the 0–3mm root apex region. Whole roots of 2-day-old etiolated maize seedlings were immersed in mock [10mM KPB (pH 6.8)] or inhibitor solutions for 1h. The following inhibitors were prepared in 10mM KBP: 50 µM yucasin+10 µM Kyn, 50 µM BFA, 100 µM NPA, and 50 µM 1-NOA+50 µM 7B-3. Roots were then placed on wet filter paper moistened with inhibitor solution, wrapped in plastic film, and incubated in darkness or under white light for 1.5h. Root tips (0–3mm, n=4–6) were collected for each treatment to quantify IAA. Data were obtained for at least two independent experiments. Values are presented as the mean ±SE (5≤n≤10). Asterisks indicate significant differences between roots treated in darkness and light-irradiated roots (Student’s t-test; **P<0.01). Plus symbols indicate significant differences between the mock control and inhibitor treatments (+P<0.05; +++P<0.001).

Fig. 5.

Incorporation of a stable isotope label into IAA after uptake of [¹³C₁₁ ¹⁵N₂]tryptophan in the root tip. Maize root tips (0–5mm region) were immersed in mock (10mM KPB) or inhibitor solutions for 30min. The following inhibitors were prepared in KPB: 50 µM yucasin+10 µM Kyn, 1mM [¹³C₁₁ ¹⁵N₂]tryptophan, and 50 µM yucasin+10 µM Kyn+1mM [¹³C₁₁ ¹⁵N₂]tryptophan. Root tips (0–10mm region) were then collected (two per treatment) to quantify IAA. Grey bar, endogenous IAA; black bar, ¹³C₉ ¹⁵N₁-labelled IAA. Values are presented as the mean ±SE (n=5 or 6). Plus symbols indicate significant differences between the mock control and yucasin+Kyn treatments (Student’s t-test; ++P<0.01).

Fig. 6.

Transcript profiles of Zmtat and Zmyuc genes in maize roots. (A) Transcription levels of Zmtat and Zmyuc genes in the 0–15mm root tip region. Approximately 200ng of total RNA was used for the RT–PCR. Ubi, Zmubiquitin. (B) Transcription of two Zmtat and five Zmyuc genes was analysed by RT–PCR. Total RNA was obtained from the 0–5, 5–10, and 10–15mm root regions. Approximately 50ng of total RNA was used for the RT–PCR. (C) Total RNA was extracted from the 0–1, 1–3, and 3–5mm root tip regions and used for qPCR. Zmubiquitin served as the internal standard. Values are presented as the mean ±SE (3≤n≤5). (D) Effects of light irradiation on the transcription of Zmvt2 and Zmyuc genes. Zmubiquitin served as the internal standard for qPCR. Transcript levels of samples treated in darkness were set to 1 and the relative transcript levels for samples exposed to light are presented. Asterisks indicate significant differences between roots treated in darkness and light-irradiated roots (Student’s t-test; *P<0.05; **P<0.01).

Fig. 7.

Effects of several IAA-related inhibitors on root U-turn behaviour and growth. (A) Whole roots of 2-day-old maize seedlings were pre-treated with inhibitors for 1h as described in the legend of Fig. 4. Roots were then inserted into capillary tubes, inverted, and incubated for 18h in darkness or under white light. For each treatment, at least three independent experiments with 4–6 seedlings were completed. Images and movies of U-turn behaviour are provided in the Supplementary data. (B) Growth rates of maize roots presented in (A). Data were obtained from two or three independent experiments (each with 9–11 roots). Black column, in darkness; white column, under white light. Values are presented as the mean ±SE. Asterisks indicate significant differences between roots treated in darkness and under light (Student’s t-test; P=0.00006). Plus symbols indicate significant differences between the mock control and chemical treatments (Student’s t-test; +P<0.05; ++P<0.01; +++P<0.001). (C) Effects of co-treatment with IAA and yucasin+Kyn on root U-turn behaviour. Samples were treated as described in (A). (This figure is available in colour at JXB online.)