YUCCA9-Mediated Auxin Biosynthesis and Polar Auxin Transport Synergistically Regulate Regeneration of Root Systems Following Root Cutting

Abstract

Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.

Keywords: Auxin biosynthesis; Lateral root; Polar auxin transport; Root pruning; YUCCA9; Yucasin.

Fig. 1

Root cutting-induced increase in lateral root (LR) number (RCN) and root cutting-induced increase in LR growth in wild-type (WT) plants. (A) Four-day-old plants were transferred to new medium and incubated for 1 d before root cutting. Photographs were taken 4 d after root cutting. Scale bars = 1 cm. Red arrowheads indicate the point 12 mm from the root–shoot junction that corresponds to the cut point. (B) The number of LRs was counted in the 12 mm area from the root–shoot junction. (C) Growth rate of the first emerged LR. (D) Total length of the root system of intact or root-cut plants 4 d after root cutting was calculated as the sum of the length of the primary root and every LR. (E) Progress of LR development after root cutting. The roots of 5-day-old plants were cut at 0 h. The number of lateral root primordia (LRPs) or LRs was counted at the indicated time points, and the sum of the LRP and LR number was defined as the total. (F) LR number within 0–6 or 6–12 mm of the cut end. (G) Histogram of LR distribution after root cutting (adventitious roots at the root–shoot junction are not included), n = 100. Error bars indicate the SE (n = 16). *Significant differences between root-cut and intact plants (Student’s t-test, P < 0.05) (B, C, F) or (χ² test, P < 0.05) (G).

Fig. 2

Root cutting induced LR formation in auxin-related mutants. Four-day-old plants were transferred to new medium and incubated for 1 d before root cutting. After root cutting, plants were incubated at control temperature (23°C) (A–E) or high temperature (28°C) (F). Photographs were taken at 4 (A–C, E left, F left) or 16 (E right, F right) days after root cutting (DAC). Scale bars = 1 cm. Red arrowheads indicate the point 12 mm from the root–shoot junction that corresponds to the cut point. (D) The number of LRs was counted in the 12 mm area from the root–shoot junction. Error bars indicate the SE (n = 16). *Significant differences between root-cut and intact plants (Student’s t-test, P < 0.05).

Fig. 3

Auxin signaling genes are induced by root cutting. (A–C) Relative expression of IAA19, ARF19 and LBD29 after root cutting. Error bars indicate the SE of three independent biological replicates. (D) The expression pattern of IAA19. The roots of 6-day-old seedlings expressing pIAA19::GUS were cut at 12 mm from the root–shoot junction. After 6 h, β-glucuronidase (GUS) staining was performed. The expression of pIAA19::GUS was observed in the cut end of root-cut plants and the corresponding area in intact plants. Scale bar = 0.1 mm.

Fig. 4

RCN is robust to auxin transport inhibitors but not to auxin biosynthesis inhibitors. Four-day-old plants were transferred to medium with or without the auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) (A), 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid (BUM) (B) or 2,3,5-triiodobenzoic acid (TIBA) (C) or the auxin biosynthesis inhibitor l-kynurenine (D) and incubated for 1 d before root cutting. The number of LRs was counted in the 12 mm area from the root–shoot junction 4 d after root cutting. (E) WT and sav3-2 plants were treated with different concentrations of yucasin. (F) The combination of yucasin and NPA abolished LR formation in both intact and cut plants. Error bars indicate the SE (n = 16). *Significant differences in root-cut vs. intact plants (A–E) and treated vs. control plants (F) (Student’s t-test, P < 0.05).

Fig. 5

IAA is induced by root cutting. Auxin concentration was measured after root cutting at the indicated time points. Error bars indicate the SE of three independent biological replicates. *Significant differences compared with 0 h (Student’s t-test, P < 0.05).

Fig. 6

The role of YUCCA9 (YUC9) following root cutting. Four-day-old WT (A) and yuc9 (B) plants were transferred to medium with or without NPA and incubated for 1 d before root cutting. Photographs were taken 4 d after root cutting. Scale bars = 1 cm. Red arrowheads indicate the point 12 mm from the root–shoot junction that corresponds to the cut point. (C) LR number of the WT and yuc9 following root cutting in the presence or absence of NPA. LR number was counted within 12 mm from the root–shoot junction 4 d after root cutting. (D) Relative expression level of YUC9 after root cutting. (E) IAA level of the WT and yuc9 at the indicated time points. (F) Progress of LR development after root cutting. The roots of 5-day-old plants were cut at 0 h. The number of LRPs or LRs was counted and the sum of LRP and LR number was defined as the total. (G) LR number of msg2-1 and msg2-1 yuc9 following root cutting in the presence or absence of NPA. LR number was counted within 12 mm from the root–shoot junction 4 d after root cutting. Error bars indicate the SE from 16 seedlings (C, F, G) or from three independent biological replicates (D, E). *Significant differences compared with WT plants (C, F), msg2-1 (G) or 0 h (E) (Student’s t-test, P < 0.05).

Fig. 7

Functional redundancy of YUC family genes in RCN. (A) Increases in LR number under different concentration of yucasin in the presence of NPA. Increases in the LR number were calculated by subtracting the LR number of intact plants from those of root-cut plants. (B) Four-day-old plants were transferred to medium with or without NPA and incubated for 1 d before root cutting. Photographs were taken 4 d after root cutting. Scale bars = 1 cm. Red arrowheads indicate the 12 mm point from the root–shoot junction that corresponds to the cut point. LR number of plants after root cutting in the absence (C) or presence (D) of NPA. LR number was counted within 12 mm from the root–shoot junction 4 d after root cutting. Scale bars = 1 cm. Error bars indicate the SE (n = 16). *Significant differences compared with the WT (Student’s t-test, P < 0.01).

Fig. 8

Synergystic effect of auxin biosynthesis and polar auxin transport (PAT) on RCN. (A) The number of LRs was examined in PAT-related mutants with or without yucasin treatment. Reduction of LR number in different concentration of the auxin transport inhibitors NPA (B) and TIBA (C). The number of LRs was counted in the 12 mm area from the root–shoot junction 4 d after root cutting. Error bars indicate the SE (n = 16). *Significant differences compared with plants in control medium (Student’s t-test, P < 0.05).

Fig. 9

Model of the synergistic regulation of RCN by auxin biosynthesis and polar auxin transport. Root cutting activates the expression of YUC9 and other YUC family genes, resulting in the elevation of the auxin level, which further induces PAT-related gene expression. Enhanced PAT activity leads to auxin accumulation and activation of downstream auxin signaling pathways which induces LR initiation and LR development.