The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutantein2-1, and auxin influx mutants aux1-7,aux1-22, and double mutant aux1-7 ein2. β-Glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains ofPS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nm) restored the root hair length ofaux1 mutants to wild-type level, whereas 100 nm NAA was needed for ein2-1 andaux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type andein2-1 roots, while stimulating these traits inaux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.

p-Chlorophenoxyisobutyric acid (PCIB) is known as a putative antiauxin and is widely used to inhibit auxin action, although the mechanism of PCIB-mediated inhibition of auxin action is not characterized very well at the molecular level. In the present work, we showed that PCIB inhibited BA::β-glucuronidase (GUS) expression induced by indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid. PCIB also inhibited auxin-dependent DR5::GUS expression. RNA hybridization and quantitative reverse transcriptase-polymerase chain reaction analyses suggested that PCIB reduced auxin-induced accumulation of transcripts of Aux/IAA genes. In addition, PCIB relieved the reduction of GUS activity in HS::AXR3NT-GUS transgenic line in which auxin inhibits GUS activity by promoting degradation of the AXR3NT-GUS fusion protein. Physiological analysis revealed that PCIB inhibited lateral root production, gravitropic response of roots, and growth of primary roots. These results suggest that PCIB impairs auxin-signaling pathway by regulating Aux/IAA protein stability and thereby affects the auxin-regulated Arabidopsis root physiology.

An Arabidopsis transgenic line was constructed expressing β-glucuronidase (GUS) via the auxin-responsive domains (AuxRDs) A and B (BA–GUS) of the PS-IAA4/5 gene in an indoleacetic acid (IAA)–dependent fashion. GUS expression was preferentially enhanced in the root elongation zone after treatment of young seedlings with 10⁻⁷ M IAA. Expression of the BA–GUS gene in the axr1, axr4, and aux1 mutants required 10- to 100-fold higher auxin concentration than that in the wild-type background. GUS expression was nil in the axr2 and axr3 mutants. The transgene was used to isolate mutants exhibiting altered auxin-responsive gene expression (age). Two mutants, age1 and age2, were isolated and characterized. age1 showed enhanced sensitivity to IAA, with strong GUS expression localized in the root elongation zone in the presence of 10⁻⁸ M IAA. In contrast, age2 exhibited ectopic GUS expression associated with the root vascular tissue, even in the absence of exogenous IAA. Morphological and molecular analyses indicated that the age1 and age2 alleles are involved in the regulation of gene expression in response to IAA.

We investigated the role of ethylene and auxin in regulating the growth and morphology of roots during mechanical impedance by developing a new growing system and using the model plant Arabidopsis (Arabidopsis thaliana). The Arabidopsis seedlings grown horizontally on a dialysis membrane-covered agar plate encountered adequate mechanical impedance as the roots showed characteristic ethylene phenotypes: 2-fold reduction in root growth, increase in root diameter, decrease in cell elongation, and ectopic root hair formation. The root phenotype characterization of various mutants having altered response to ethylene biosynthesis or signaling, the effect of ethylene inhibitors on mechanically impeded roots, and transcription profiling of the ethylene-responsive genes led us to conclude that enhanced ethylene response plays a primary role in changing root morphology and development during mechanical impedance. Further, the differential sensitivity of horizontally and vertically grown roots toward exogenous ethylene suggested that ethylene signaling plays a critical role in enhancing the ethylene response. We subsequently demonstrated that the enhanced ethylene response also affects the auxin response in roots. Taken together, our results provide a new insight into the role of ethylene in changing root morphology during mechanical impedance.

Gamma rays are the most frequently used ionizing radiation in plant mutagenesis; however, few studies are available on the characteristics of mutations at a genome-wide level. Here, we quantitatively and qualitatively characterized the mutations induced by acute/chronic gamma ray irradiation in Arabidopsis. The data were then compared with those previously obtained for carbon ion irradiation. In the acute irradiation of dry seeds at the same effective survival dose, gamma rays and carbon ions differed substantially, with the former inducing a significantly greater number of total mutation events, while the number of gene-affecting mutation events did not differ between the treatments. This may result from the gamma rays predominantly inducing single-base substitutions, while carbon ions frequently induced deletions ≥2 bp. Mutation accumulation lines prepared by chronic gamma irradiation with 100–500 mGy/h in five successive generations showed higher mutation frequencies per dose compared with acute irradiation of dry seeds. Chronic gamma ray irradiation may induce larger genetic changes compared with acute gamma ray irradiation. In addition, the transition/transversion ratio decreased as the dose rate increased, suggesting that plants responded to very low dose rates of gamma rays (∼1 mGy/h), even though the overall mutation frequency did not increase. These data will aid our understanding of the effects of radiation types and be useful in selecting suitable radiation treatments for mutagenesis.

Ionizing radiation including heavy-ion beams has been widely used in mutation breeding. Dry seeds, seedlings, and cultured tissues are often used for mutagenesis; however, little is known about the differences in induced mutations among them. Here, we examined the characteristics of mutations using randomly chosen Arabidopsis M₂ plants derived from dry seeds and seedlings irradiated with carbon ions. The mutation frequency was 1.4–1.9 times higher in dry-seed irradiation than in seedling irradiation. This difference was mainly due to the three-times higher frequency of insertions and deletions (InDels) in dry-seed irradiation than in seedling irradiation. This difference increased the proportion of mutations predicted to affect gene function among all mutations identified by whole genome re-sequencing. Our results demonstrate that the physiological status of plant tissue greatly affects the characteristics of mutations induced by ionizing radiation, and that dry seeds are more suitable materials than seedlings for inducing loss-of-function mutations. The results also showed that single base deletions often occurred in homopolymeric sequences, while InDels larger than 2–3 bp often occurred in or near polynucleotide-repeat or microhomologous sequences. Interestingly, microhomology was less commonly found around large deletions (≥50 bp), suggesting that the rejoining process differs depending on the deletion size.