Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana - PubMed (original) (raw)

Ethylene regulates lateral root formation and auxin transport in Arabidopsis thaliana

Sangeeta Negi et al. Plant J. 2008 Jul.

Abstract

Lateral root branching is a genetically defined and environmentally regulated process. Auxin is required for lateral root formation, and mutants that are altered in auxin synthesis, transport or signaling often have lateral root defects. Crosstalk between auxin and ethylene in root elongation has been demonstrated, but interactions between these hormones in the regulation of Arabidopsis lateral root formation are not well characterized. This study utilized Arabidopsis mutants altered in ethylene signaling and synthesis to explore the role of ethylene in lateral root formation. We find that enhanced ethylene synthesis or signaling, through the eto1-1 and ctr1-1 mutations, or through the application of 1-aminocyclopropane-1-carboxylic acid (ACC), negatively impacts lateral root formation, and is reversible by treatment with the ethylene antagonist, silver nitrate. In contrast, mutations that block ethylene responses, etr1-3 and ein2-5, enhance root formation and render it insensitive to the effect of ACC, even though these mutants have reduced root elongation at high ACC doses. ACC treatments or the eto1-1 mutation significantly enhance radiolabeled indole-3-acetic acid (IAA) transport in both the acropetal and the basipetal directions. ein2-5 and etr1-3 have less acropetal IAA transport, and transport is no longer regulated by ACC. DR5-GUS reporter expression is also altered by ACC treatment, which is consistent with transport differences. The aux1-7 mutant, which has a defect in an IAA influx protein, is insensitive to the ethylene inhibition of root formation. aux1-7 also has ACC-insensitive acropetal and basipetal IAA transport, as well as altered DR5-GUS expression, which is consistent with ethylene altering AUX1-mediated IAA uptake, and thereby blocking lateral root formation.

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Figures

Figure 1

Figure 1

The root branching patterns of Arabidopsis are influenced by mutations that alter ethylene signaling and synthesis. (a) Roots 10 days after sowing are shown. (b) The number of emerged lateral roots in each genotype at 10 days after sowing. The average and SE of 30 seedlings is reported. (c) Images of roots that were grown for 5 days after sowing, before being transferred to control media or media containing 10 μ

m

silver nitrate, and photographed after five additional days of growth. P < 0.0005 by a Student’s _t_-test, as compared with untreated Col. Scale bars: 5 mm.

Figure 2

Figure 2

1-Aminocyclopropane-1-carboxylic acid (ACC) reduces root initiation in Col, but not in ethylene-insensitive mutants. Seedlings were grown on control media for 5 days and were then transferred to 1 μ

m

ACC or the indicated concentration, or 10 μ

m

silver nitrate, for five additional days. (a) Lateral root phenotypes are shown with a scale bar of 5 mm. (b and c) The effects of a range of ACC concentrations on the number of lateral roots or the root length were determined, with the average and SE of 30 seedlings reported here (d) The average number of lateral roots and SE of 30 Col seedlings for each treatment are shown with significant differences (P < 0.01) compared with untreated seedlings, indicated by the letter ‘a’, or with and without silver treatment, indicated by ‘b’, and silver treatment with or without ACC, indicated by ‘c’ (e) The number of lateral root early primordia, emerged lateral roots, and the combined totals were determined by CYCB1;1:GUS expression. The average and SE of 20 seedlings are reported, and significant differences between treatments and within stages were determined by a Student’s _t_-test, P< 0.001.

Figure 3

Figure 3

Acropetal indole-3-acetic acid (IAA) transport is positively correlated with ethylene levels and responses. Seedlings were grown on control media for 5 days, and were then transferred to different treatments for 24 h. The average and SE of 30 seedlings are reported in all panels, and statistical analysis was performed using the Student’s _t-_test with significant differences relative to untreated Col indicated; *P < 0.05. (b–d) Tritiated IAA transport was quantified in the apical 5 mm of the root tip. (a) Acropetal transport in several genotypes are compared measuring the radioactivity in three 5-mm segments: upper (near the root shoot junction), middle and tip (b) Col seedlings were treated with 1 μ

m

1-aminocyclopropane-1-carboxylic acid (ACC) and/or 10 μ

m

silver nitrate (c) Col and eto1-1 seedlings were treated with 10 μ

m

silver nitrate (d) Col, ein2-5 and etr1-3 seedlings were treated with 1 μ

m

ACC (e) DR5:GUS expression in roots treated with 1 μ

m

ACC and/or 10 μ

m

silver nitrate. Scale bar: 40 μm.

Figure 4

Figure 4. Basipetal indole-3-acetic acid (IAA) transport is increased with 1-aminocyclopropane-1-carboxylic acid (ACC) treatment and in eto1-1 seedlings

Five-day-old Col or eto1-1 seedlings were treated with 1 μ

m

ACC, 10 μ

m

silver nitrate or both, and basipetal IAA transport was quantified after 24 h of treatment. The average and SE of 30 seedlings are reported, and statistical analysis was performed using the Student’s _t-_test, with all values being significant with P < 0.0001, with the following comparisons within genotypes: a, with and without ACC; b, with and without silver nitrate; c, with ACC and with and without silver nitrate, and between genotypes; d, untreated Col versus silver nitrate treated eto1; and e, untreated Col versus eto1-1.

Figure 5

Figure 5

Ethylene-insensitive mutants are less sensitive to naphthylphthalamic acid (NPA). Col, ein2-5 and etr1-3 seedlings were grown on control media, and after 5 days were transferred to media containing the indicated doses of NPA. After 5 days of growth in the presence of NPA, the number of emerged lateral roots was quantified. Data are presented as a percentage of lateral roots in the untreated control. The average and SE of 20 seedlings are reported.

Figure 6

Figure 6

Exogenous indole-3-acetic acid (IAA) can reverse the negative effect of 1-aminocyclopropane-1-carboxylic acid (ACC) on lateral root formation. Col, ein2-5 and eto1-1 seedlings were grown on control media, and after 5 days were transferred to media containing 1 μ

m

ACC, 1 μ

m

IAA or both. (a) At the time of transfer, the plate was marked with a dot at the position of the root tip. After 5 days of growth in the presence of the indicated treatments, images of roots were captured. (b) The number of lateral roots was quantified. The average and SE of 30 seedlings and significant differences within genotypes are reported; *P< 0.001.

Figure 7

Figure 7

Lateral root formation and indole-3-acetic acid (IAA) transport are less ethylene-sensitive in aux1-7 (a) Col and aux1-7 were transferred to media containing 1 μ

m

1-aminocyclopropane-1-carboxylic acid (ACC) at 5 days after sowing, and the number of emerged lateral roots was quantified after five additional days (b) The effect of ACC on acropetal IAA transport in Col and aux1-7 are compared (c) The effect of 1 μ

m

ACC on basipetal transport in aux1-7 and Col are compared (d) _DR5:_GUS expression is shown in Col and aux1-7 in the presence and absence of 1 μ

m

ACC; the scale bar is 40 μm. For all experiments, the average and SE of 20–30 seedlings, and significant differences, are reported for comparisons between untreated genotypes or for treatment within genotypes; *P< 0.001.

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