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. 2013 Oct;112(6):1107-16.
doi: 10.1093/aob/mct181. Epub 2013 Aug 21.

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Maria G Ivanchenko  1 , Désirée den Os, Gabriele B Monshausen, Joseph G Dubrovsky, Andrea Bednárová, Natraj Krishnan
Affiliations

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Maria G Ivanchenko et al. Ann Bot. 2013 Oct.

Abstract

Background and aims: The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods: Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum 'Ailsa Craig') were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H2O2), and expression of ROS-related mRNAs.

Key results: The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H2O2) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H2O2 concentration in the root tip in a dose-dependent manner. Auxin and H2O2 elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H2O2 and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions: The results indicate that auxin regulates the level of H2O2 in the root tip, so increasing the auxin level triggers accumulation of H2O2 leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H2O2 homeostasis in the root tip.

Keywords: Auxin; ROS; Solanum lycopersicum; dgt; diageotropica; hydrogen peroxide; root elongation; tomato.

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Figures

Fig. 1.
Fig. 1.
The dgt mutant of tomato displays reduced root growth correlated with reduced meristem length, number of cells in the elongation zone and elongated cell length. (A) Images of 8-d-old wild-type and dgt seedlings. Two partially overlapping images were combined for each genotype by precisely aligning the contours of overlapping cells. (B) Root meristem in wild-type and dgt depicted as the distance between the QC/root cap border and the beginning of cell elongation (dashed lines). Inserts show beginning of cell elongation in the second exterior layer of the cortex. Scale bar = 122 µm. (C) Expression level and IAA response of Sl-CyclinB1;1 cell-cycle gene in wild-type and dgt root tips. Different letters indicate a statistically significant difference when analysed by one-way ANOVA and a multiple comparison using Tukey's test at P ≤ 0·05. Data are combined mean ± s.e. of two experiments performed in triplicate with 50–100 root tips per sample. (D) Quantifications of root meristem length, meristem cell number, elongation zone (EZ) length, elongation zone cell number and elongated root cell length in wild-type and dgt. Quantitative values are combined mean ± s.e. for two independent experiments with 11–12 roots per sample in each experiment. *P ≤ 0·05, Student's t-test.
Fig. 2.
Fig. 2.
Histological comparison of the distal meristem region from a wild-type and dgt root. The QC cell region is marked. Periodic acid-Schiff stain was used to visualize cells in 8-d-old seedlings. Scale bar = 35 µm.
Fig. 3.
Fig. 3.
The dgt mutation increases the H2O2 level in the root tip but has no visible effect on the O2•– level. (A) Images of 8-d-old wild-type roots stained for the superoxide radical (O2•–) using Nitroblue tetrazolium (NBT) and for H2O2 using 3,3′-diaminobenzidine tetrachloride (DAB), respectively. Note that the NBT signal increases toward the root tip whereas the DAB signal increases in the opposite direction (arrows). (B) Comparison of wild-type and dgt root tips stained for O2•–. (C) Comparison of wild-type and dgt root tips stained for H2O2. Scale bars = 122 µm.
Fig. 4.
Fig. 4.
Confocal imaging of extracellular ROS accumulation along wild-type and dgt tomato roots incubated with OxyBURST green H2HFF-BSA. (A) Bright field (left) and fluorescence images (right) of growing wild-type and dgt root tips 1 min after addition of OxyBURST. Regions (–3) spanning the meristem and the elongation zone used to measure fluorescence are marked. The beginning of root hair initiation is marked in dgt (arrow) and is just above the field of the view for wild-type. Fluorescence quickly developed along the root surface of dgt, indicating higher extracellular ROS accumulation compared with wild-type. Scale bar = 500 µm. (B) Quantification of ROS-dependent OxyBURST fluorescence in region 1 (black), region 2 (red) and region 3 (blue) as denoted in A. Average ROS-dependent fluorescence intensities were analysed by three-way ANOVA followed by Tukey's HSD, which showed a statistically significantly difference between wild-type and dgt (P < 0·05 for regions 1 and 2, P < 0·01 for region 3). Within wild-type there was no significant difference between regions or over time, while in dgt a significant difference was recorded between region 1 and 3 (P < 0·01) and between 2 and 3 (P < 0·05). Data represent means ± s.d. of six independent measurements. a.u., arbitrary units.
Fig. 5.
Fig. 5.
Correlations between IAA level and response and H2O2 level and response in tomato seedlings. (A) Quantifications of the effect of IAA on H2O2 level in root tips of 8-d-old wild-type and dgt seedlings that had received a 12-h IAA treatment. (B) Comparisons of the effects of IAA and H2O2 on root growth, meristem length, elongation zone (EZ) length, elongation-zone cell number, and elongated cell length in 8-d-old wild-type and dgt seedlings after 5 h of treatment. Different letters indicate a statistically significant difference between treatments in each genotype as well as between genotypes at P ≤ 0·05 when analysed by two-way ANOVA followed by Bonferroni's post-test. Data in A represent mean ± s.e.m. from three independent experiments (n = 3) where each sample consists of 50–100 pooled root tips, and data in B represent mean ± s.e.m. of two independent experiments each performed with 10–11 individual roots per group (n = 20–22).
Fig. 6.
Fig. 6.
Quantifications of the effect of IAA (as indicated in the key) on expression level of mRNAs of tomato ROS-scavenging enzymes (Sl-SOD accession M37151, Sl-CAT1 accession M93719, Sl-APX1 accession DQ099420, Sl-GPXL accession NM_001247638, Sl-GST/GPX accession NM_001247450), mRNAs related to regulation of antioxidant balance (Sl-GCS accession NM_001247081, Sl-GR accession FJ265823, Sl-AO accession AY971876, Sl-DHAR accession AY971873) and DGT mRNA (accession M55019). Different letters indicate a statistically significant difference at P ≤ 0·05 when analysed by two-way ANOVA followed by Bonferroni's post-test. Data represent combined mean ± s.e. of three biological experiments performed in triplicate (n = 9) with 50–100 pooled root tips per sample.
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