Asymmetric auxin distribution establishes a contrasting pattern of aerenchyma formation in the nodal roots of Zea nicaraguensis during gravistimulation

Abstract

Auxin distribution is essential for determining root developmental patterns. The formation of lateral roots and constitutive aerenchyma, which is a gas space developed through cell death, is regulated by auxin in rice (Oryza sativa). However, it is unclear whether the involvement of auxin in constitutive aerenchyma formation is conserved in other species. In this study, we found that constitutive aerenchyma formation was regulated by auxin in the nodal roots of Zea nicaraguensis, a wild relative of maize (Zea mays ssp. mays) grown naturally on frequently flooded coastal plains. Subsequent gravistimulation (root rotation) experiments showed opposite patterns of aerenchyma and lateral root formation. Lateral root formation on the convex side of rotated roots is known to be stimulated by a transient increase in auxin level in the pericycle. We found that aerenchyma formation was accelerated in the cortex on the concave side of the rotated nodal roots of Z. nicaraguensis. A cortex-specific expression analysis of auxin-responsive genes suggested that the auxin level was higher on the concave side than on the convex side. These results suggest that asymmetric auxin distribution underlies the regulation of aerenchyma and lateral root formation in the nodal roots of Z. nicaraguensis. As aerenchyma reduces the respiratory cost of the roots, constitutive aerenchyma on the concave side of the nodal root may balance resource allocation, thereby contributing to the uptake of water and nutrients by newly formed lateral roots. Our study provides insights into auxin-dependent asymmetric root patterning such as that of gravistimulation and hydropatterning response.

Keywords: Zea nicaraguensis; aerenchyma; auxin distribution; cell death; gravistimulation; laser microdissection; lateral root; maize (Zea mays ssp. mays).

Figure 1

Aerenchyma formation in the third nodal roots of Z. nicaraguensis under aerated conditions with IAA and/or NPA treatments. (A) Cross-sections at 40, 50, 60, 70, and 80 mm from the tips of third nodal roots with or without 5 µM IAA and 0.05 µM NPA treatments. Scale bar = 200 μm. (B) Percentage of aerenchyma in root cross-sectional areas at 30, 40, 50, 60, 70, and 80 mm from the root tips. Boxplots show the median (horizontal lines), 25^th to 75^th percentiles (edge of the boxes), minimum to maximum (edge of the whiskers), and mean values (dots in the boxes) (n = 6). Significant differences among the treatments are denoted with different lowercase letters (p < 0.05, one-way ANOVA, followed by LSD test for multiple comparisons).

Figure 2

Age-dependent aerenchyma formation in the third nodal roots of Z. nicaraguensis under aerated conditions with IAA and/or NPA treatments. (A) Elongation rate of the nodal roots of Z. nicaraguensis seedlings grown under aerated conditions with IAA and/or NPA treatments. Boxplots show the median (horizontal lines), 25^th to 75^th percentiles (edge of the boxes), minimum to maximum (edge of the whiskers), and mean values (dots in the boxes) (n = 6). Significant differences among the treatments are denoted with different lowercase letters (p < 0.05, one-way ANOVA, followed by LSD test for multiple comparisons). (B) Schematic diagram of root elongation and differences in the distances from the root tips among the treatments at 0 h and 48 h. (C) The Gompertz curve is fitted for the percentage of aerenchyma formed. The obtained equations for each of the treatments are indicated above the graph. (D) Age-dependent aerenchyma formed was predicted by the equations obtained from Gompertz curve fittings. The percentage of aerenchyma formed at each time point was calculated using the distance from the root tips at each time point as the explanatory variable, which was calculated from the elongation rate under each treatment (A).

Figure 3

Lateral root formation on the concave and convex sides of the gravity-stimulated nodal roots of Z. nicaraguensis. (A) Diagram of gravistimulation experiment. The arrow indicates the direction of gravity (g) during the control and rotated treatments. (B) Pictures of lateral root primordia in the control and rotated nodal roots in the bent region (0 mm ± 2 mm) and at 10 mm (± 2 mm) from the bending position (at 0 mm). The basal and apical directions from the upright position are indicated with minus (−) and plus (+) signs, respectively. LR primordia were visualized using DAPI staining. Scale bars = 1 mm. Numbers of lateral root primordia at −10, 0, and +10 mm on the concave side (C) and convex side (D) and the total number of lateral root primordia in the nodal roots (E). Significant differences between the control and rotated nodal roots at p < 0.05 (two-sample t-test) are denoted with asterisks. Values are mean ± SD (n = 3 to 4).

Figure 4

Aerenchyma formation on the concave and convex sides of the gravity-stimulated nodal roots of Z. nicaraguensis. (A) Cross-sections of the control and rotated nodal roots in the bent region (0 mm ± 2 mm) and at 10 mm (± 2 mm) from the bending position (at 0 mm). The parts cut by a razor blade are indicated with arrowheads. Scale bar = 1 mm. The percentage of aerenchyma formed at −10, 0, and +10 mm on the concave side (B) and convex side (C) and the total percentage of aerenchyma formed in the nodal roots (D). Significant differences between the control and rotated nodal roots at p < 0.05 (two-sample t-test) are denoted with asterisks. Values are mean ± SD (n = 6).

Figure 5

Time-course of aerenchyma formation in the cortex on the concave and convex sides in the bent region (0 mm ± 2 mm) of the gravity-stimulated nodal roots of Z. nicaraguensis. Percentage of aerenchyma formed on the concave side (A) and convex side (B) of the nodal roots at 24, 48, and 72 h after the initiation of the treatment. Boxplots show the median (horizontal lines), 25^th to 75^th percentiles (edge of the boxes), minimum to maximum (edge of the whiskers), and mean values (dots in the boxes) (n = 5). Significant differences between the control and rotated nodal roots at p < 0.05 (two-sample t-test) are denoted with asterisks.

Figure 6

Time-course expression levels of the auxin-responsive genes in the cortex on the concave and convex sides in the bent region (0 mm ± 2 mm) of the gravity-stimulated nodal roots of Z. nicaraguensis. Pictures of the longitudinal section of the nodal roots before (A) and after (B) the isolation of the cortex using laser microdissection. Scale bar = 300 µm. Copy numbers of the transcripts of AAS2 (C), RUM1 (D), SAUR6 (E), and SAUR24 (F) in the cortex on the concave and convex sides at 12, 24, and 36 h after the gravistimulation treatment. Significant differences between the concave and convex sides at each time point are denoted with different lowercase letters (p < 0.05, one-way ANOVA, followed by LSD test for multiple comparisons). Values are mean ± SD (n = 3 to 5).

Figure 7

Model of contrasting aerenchyma formation regulated by asymmetric auxin distribution in the cortex of the gravity-stimulated nodal roots of Z. nicaraguensis. (A) Lateral root (LR) and constitutive aerenchyma (CA) formation is symmetric in the control. In the cortex, longitudinal gradients of auxin stimulate CA formation. (B) LR and CA formations are asymmetric under the gravistimulation treatment. Auxin levels increase and decrease in the cortex on the concave and convex sides, respectively. Thus, CA formation is accelerated on the concave side and retarded on the convex side of the nodal roots. By contrast, a transient increase in the auxin level on the convex side of the pericycle promotes LR formation. C; concave side, V; convex side.