Manipulating Ethylene Synthesis in Arabidopsis Seedlings Also Alters the Synthesis and Abundance of Auxin.
(A) IAA synthesis and abundance was measured in 6-d-old Arabidopsis seedlings that were incubated for 12 and 24 h with D₂O medium containing the ethylene precursor ACC (top panel) or the ethylene synthesis inhibitor AVG (middle panel). IAA abundance was measured in 6-d-old Arabidopsis seedlings that were incubated for 12 and 24 h with D₂O medium containing AVG (bottom panel). FW, fresh weight.
(B) IAA synthesis was measured in 6-d-old Arabidopsis seedling roots treated with the ethylene synthesis inhibitor AVG. After 6 d of growth, either whole seedlings (top panel) or excised roots (bottom panel) were fed with D₂O medium containing AVG for 24 h. IAA synthesis was measured in 2-mm segments of wild-type Arabidopsis roots sampled from tissues 0 to 2 mm and 2 to 4 mm from the apex. After correction for natural isotope abundances, ratios between the labeled tracer (mass-to-charge ratio [m/z] of 203, 204, and 205) and unlabeled tracee (m/z 202) were calculated for IAA (expressed as the t/t-ratio).

aux1 Blocks the Induction of Auxin (but Not Ethylene) Responsive Gene Expression after Ethylene Treatment.
Transcript abundance of the auxin-responsive gene IAA1 (Abel and Theologis, 1995) and ethylene-responsive gene ERF1 (Solano et al., 1998) was measured employing qRT-PCR. RNA was extracted from 10-d-old wild-type and aux1-22 seedling roots 3, 6, and 15 h after treatment with ethylene (2 ppm) or ethylene-free air (controls). Data represent the average of three biological repeats (±se). All values are normalized to that of Col-0 exposed to ethylene-free air for the same time period.

Induction of the Auxin-Responsive Reporter IAA2_pro:GUS by Ethylene Is Dependent on EIN2 and AUX1.
The expression of the IAA2_pro:GUS reporter was monitored in the wild type ([A] and [F]), in the constitutive ethylene response mutant ctr1 ([B] and [G]), in the ethylene-insensitive mutant ein2 ([C] and [H]), in the auxin influx carrier null allele aux1-22 ([D] and [I]), and in the partial loss-of-function allele aux1-2 ([E] and [J]). Seedlings were either grown in air ([A] to [E]) or in the presence of 10 μL/L of ethylene ([F] to [J]).

Inhibition of Root Cell Elongation by ACC Is Dependent on AUX1.
Profiles of cortical ([A] and [B]), epidermal trichoblast ([C] and [D]), and atrichoblast ([E] and [F]) cell expansion in wild-type (circles), aux1 (diamonds), and aux1 J0951≫AUX1 (triangles) roots. Seedlings were either grown in the absence ([A], [C], and [E]) or in the presence ([B], [D], and [F]) of 1 μM ACC. Cell lengths were measured relative to the quiescent center (QC) at the root apex using confocal microscopy (see Methods).

Kinematic Analysis of Root Growth Reveals That ACC Inhibits Rapid Cell Expansion.
Profiles of velocity ([A] and [B]) and relative rates of cell expansion ([C] and [D]) along the root elongation zones of the wild type (circles), aux1 (diamonds), and aux1 J0951≫AUX1 (triangles) were obtained from kinematic analysis. Seedlings were grown either in the absence ([A] and [C]) or in the presence ([B] and [D]) of 1 μM ACC.

Ethylene-Inhibited Root Growth Is Dependent on Auxin Transport and Responses.
(A) Schematic diagram summarizing Arabidopsis root apical tissue organization. LRC, lateral root cap; QC, quiescent center.
(B) The sensitivity of aux1 root growth toward ethylene could be restored by targeting the expression of AUX1 in elongating epidermal and/or the lateral root cap cells in the aux1 J0951≫AUX1, aux1 Q1220≫AUX1, and aux1 M0013≫AUX1 lines, respectively.
(C) The sensitivity of wild-type root growth toward ethylene could be partially disrupted by targeting the expression of axr3-1 in cortical/endodermal or epidermal cells in the J0951≫axr3-1 and J0571≫axr3-1 lines, respectively. Strong ethylene resistance was observed in the J0631≫axr3-1 line, which expresses axr3-1 in every elongation zone tissue. Seedlings were grown vertically on Murashige and Skoog (MS) plates either in air or ethylene (10 μL/L) for 5 d. Root growth in the presence or absence of ethylene was measured and expressed as a percentage of root growth compared with the air control. Asterisks indicate significant difference from the control (P < 0.05; see Supplemental Figure 5 online for further details). The color coding of bars either refers to the root tissues in which either AUX1 or axr3-1 expression is targeted or, if black, denotes them as controls. The aux1-22, etr1-1, and axr3-1 mutants represent ethylene-resistant controls.

Induction of IAA2_pro:GUS by Ethylene Is Blocked by axr2-1 and axr3-1.
The expression of the IAA2_pro:GUS reporter was monitored in a variety of auxin response mutant backgrounds either grown in air ([A] to [E]) or after ethylene treatment (10 μL/L; [F] to [J]).

Model for Auxin Action during Ethylene-Regulated Root Growth.
The model provides a spatial framework on which to place auxin components required for ethylene responses in roots. The schematic diagram of the Arabidopsis root apex is overlaid with the sites of action of auxin biosynthesis (WEI2/ASA1 and WEI7/ASB1) and transport (AUX1) components required for ethylene inhibition of root growth. Small arrows denote the basipetal flow of auxin from the root apex via the lateral root cap to the elongation zone. Sites of ethylene-responsive gene expression are denoted by large horizontal arrows. We also highlight that the dominant gain-of-function mutation axr3-1/iaa17 can disrupt ethylene inhibition of root growth in elongation zone tissues. Vertical arrows either represent the time required or distance moved by root cells as they progress through meristematic and elongation zones.