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MES Buffer Affects Arabidopsis Root Apex Zonation and Root Growth by Suppressing Superoxide Generation in Root Apex

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MES Buffer Affects Arabidopsis Root Apex Zonation and Root Growth by Suppressing Superoxide Generation in Root Apex

Tomoko Kagenishi et al. Front Plant Sci.

Abstract

In plants, growth of roots and root hairs is regulated by the fine cellular control of pH and reactive oxygen species (ROS). MES, 2-(N-morpholino)ethanesulfonic acid as one of the Good's buffers has broadly been used for buffering medium, and it is thought to suit for plant growth with the concentration at 0.1% (w/v) because the buffer capacity of MES ranging pH 5.5-7.0 (for Arabidopsis, pH 5.8). However, many reports have shown that, in nature, roots require different pH values on the surface of specific root apex zones, namely meristem, transition zone, and elongation zone. Despite the fact that roots always grow on a media containing buffer molecule, little is known about impact of MES on root growth. Here, we have checked the effects of different concentrations of MES buffer using growing roots of Arabidopsis thaliana. Our results show that 1% of MES significantly inhibited root growth, the number of root hairs and length of meristem, whereas 0.1% promoted root growth and root apex area (region spanning from the root tip up to the transition zone). Furthermore, superoxide generation in root apex disappeared at 1% of MES. These results suggest that MES disturbs normal root morphogenesis by changing the ROS homeostasis in root apex.

Keywords: 2-(N-morpholino)ethanesulfonic acid; Arabidopsis; ROS; root; root hair; transition zone.

Figures

FIGURE 1
FIGURE 1
Root growth and morphology in different concentrations of MES. (A) Appearances of seedlings at 6-day old. The seeds were germinated on 1/2 MS medium containing each concentration of MES. Black marks indicate positions of root apices at 3-day old. (B) Root lengths at 3-day after germination on each concentration of MES. Error bars indicate standard deviation of the mean (n = 62–72). (C) Root lengths at 6-day after germination old on each concentration of MES. Error bars indicate standard deviation of the mean (n = 34–65). (D) The number of waves of the roots. Error bars indicate standard deviation of the mean (n = 17). (E) Root hair formation affected by MES. Lengths of root hairs were measured at position where long root hairs emerge, because the distance from the tip varies under different MES treatments. Five root hairs were measured from five seedlings. Error bars indicate standard deviation of the mean (n = 25). Different letters indicate significant difference (Tukey’s HSD test, P < 0.05).
FIGURE 2
FIGURE 2
Comparison of the root apex lengths. (A) A scheme of the root apex. Cells with asterisks indicate the typical cells in transition zone. In this study, we define the border between transition zone and elongation zone as following morphological category: the cell lengths obtain values which are two times higher as the cell widths (Baluška et al., 2010). (B) Microscope images of the root apices. Tips of black triangulates indicate positions of the end of transition zone. A dashed horizontal line indicates the position of root apices. (C) Lengths of the root apices (from the root tip to the end of the transition zone). Error bars indicate standard deviations of the mean (n = 9–11). Different letters indicate significant difference (Tukey’s HSD test, P < 0.05).
FIGURE 3
FIGURE 3
Light Stereomicroscope pictures of NBT staining for superoxide detection. Seedlings were grown in different concentrations of MES for 6 days. These were incubated for 5 min in NBT. The representative picture is shown here (n = 5).
FIGURE 4
FIGURE 4
A schematic diagram of MES effects on root growth. MES disturbs ROS-generating pathway in the root apex, possibly via enzymes (e.g., peroxidases), affecting the root apex zonation. The ROS (O2) is involved in root hair formation, root growth and root tropisms.

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