The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling

Abstract

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.

Figure 1.

The PLS Gene Regulates Ethylene Responses. (A) Representative seedlings of the wild type (C24 and Col-0 [for Columbia]), pls, eto1-1, PLS transgenic overexpresser (PLSOx), and ein2 grown in the dark in air, showing the triple-response phenotype of eto1-1 and pls and the etiolated phenotypes of the wild-type, ein2, and PLSOx seedlings. (B) Representative seedlings of the wild type (C24), etr1-1, ctr1, and pls grown in the dark in air, showing the triple-response phenotype of ctr1 and pls and the etiolated phenotypes of the wild-type and etr1-1 seedlings. (C) Top, RNA gel blot analysis showing increased accumulation of the ethylene-inducible At GSTF2 mRNA in air-grown pls seedlings compared with wild-type seedlings. Bottom, RNA loading control (ethidium bromide–stained 28S rRNA). A total of 10 μg of RNA was loaded per lane. (D) Semiquantitative RT-PCR of the ethylene-inducible ERF10 transcript in 7-d-old wild-type and pls seedlings. Amplification of ACT2 as an RNA loading control is shown. M, RNA size markers.

Figure 2.

Restoration of pls Root Growth by Inhibition of Ethylene Signaling. (A) Representative light-grown seedlings of etr1-1, pls, and the pls etr1-1 double mutant, showing rescued root growth in the double mutant. (B) Rescue of primary root growth in pls seedlings treated with 1 μM silver ions [pls (Ag)], which inhibit ethylene signaling by modifying ETR1 conformation or signal propagation to the kinase domain of the receptor (Rodriguez et al., 1999). Error bars represent se; n = 10. (C) Representative dark-grown seedlings of etr1-1, pls, and the pls etr1-1 double mutant in air, showing suppression of the triple response of pls in the double mutant.

Figure 3.

pls Is Not an Ethylene Biosynthesis Mutant. (A) Ethylene evolution by wild-type (Col and C24), pls, and eto1-4 seedlings. Error bars represent sd; n = 6. (B) Phenotypes of representative seedlings of pls, pls cin5, and cin5 mutants grown in the dark for 3 d in the presence of 0.5 μM kinetin, showing suppression of the cin5 phenotype by pls in the double mutant. (C) Effect of growth for 7 d on ACC on primary root length of dark-grown seedlings of the wild type (Col-0) and PLS open reading frame-overexpressing line 38. The first data point is 0.5 μM ACC. Error bars represent se; n = 10. (D) Phenotypes of representative seedlings of light-grown PLSOx (left), ctr1-1 (right), and PLS overexpresser carrying the ctr1-1 mutation (PLSOx/ctr1; middle). (E) Phenotypes of representative seedlings of dark-grown PLSOx (left), ctr1-1 (right), and PLSOx/ctr1 (middle). (F) Representative seedlings of dark-grown PLSOx (left) in the presence of 100 μM ACC and PLSOx/ctr1 (right) grown in air. (G) Representative seedlings of light-grown PLSOx (left) in the presence of 100 μM ACC and PLSOx/ctr1 (right) grown in 100 μM ACC.

Figure 4.

Auxin Transport and Accumulation Are Defective in pls. (A) Lateral root numbers in pls, pls etr1-1, and wild-type seedlings at 10 d after germination. Error bars represent se; n = 10. (B) Free IAA content of pls and wild-type seedlings in aerial and root tissues at 4, 7, and 10 d after germination. Error bars represent sd; n = 5. FW, fresh weight. (C) Free IAA content of wild-type, pls, pls etr1-1, and PLSOx seedlings at 10 d after germination. Error bars represent sd; n = 3. FW, fresh weight. (D) Polar transport of auxin in wild-type and pls inflorescence stems. N indicates lack of transport in the basal–to-apical direction. Error bars represent se; n = 8. (E) Auxin transport in pls and pls etr1-1 mutants. Error bars represent se; n = 7 for pls and pls etr1-1; n = 8 for the wild type.

Figure 5.

pls and ACC Suppress the rty Mutant Phenotype. (A) and (B) Seedlings of the rty single mutant and rty pls double mutant, showing adventitious root formation on the hypocotyl and primary root in rty. In the rty pls double mutant (B), the reduced frequency of lateral roots compared with rty is apparent. (C) rty seedlings treated with the ethylene precursor ACC (rty + ACC) show a reduced frequency of lateral roots. (D) Seedlings of ethylene-overproducing eto1-1, rty, and the double mutant eto1-1 rty. The double mutant shows a reduced frequency of lateral roots.

Figure 6.

pls Has Reduced Responses to Microtubule Inhibitors. (A) and (B) Wild-type, pls, etr1-1, and pls etr1-1 seedlings grown for 10 d in the presence ([A] and + in [B]) or absence (− in [B]) of 5 μM APM. (C) and (D) Wild-type, PLSOx, and ein2 seedlings grown for 10 d in the presence (+) or absence (−) of 5 μM APM. (E) Wild-type and pls seedlings grown for 10 d in the presence of 5 μM oryzalin. (F) Effects of propyzamide on the primary root phenotype of wild-type, pls, and PLSOx seedlings. (G) Kinetics of APM effects on root growth in pls and wild-type seedlings. The 50% inhibitory dose of APM is 1.5 μM for the wild type and 6.0 μM for pls. Each data point represents the mean of six measurements. (H) Kinetics of propyzamide effects on primary root growth of wild-type, pls, and PLSOx seedlings. Each data point represents the mean of six measurements.

Figure 7.

PLS Expression Is Repressed by Ethylene. (A) to (C) PLS:GUS expression in At EM101 ([A], −ACC, and [B]) is reduced on treatment with 100 μM ACC ([A], +ACC, and [C]). (D) to (F) GUS activity in wild-type seedlings transformed with P_PLS:GUS ([D], −ACC, and [E]) is reduced on treatment with 100 μM ACC ([D], +ACC, and [F]). (G) RNA-specific RT-PCR of the wild-type PLS transcript (443-bp product) in 6-d-old wild-type seedlings untreated (U), treated for 24 h with 10 μM 1-naphthylacetic acid (NAA), or treated for 24 h with 100 μM ACC. The –RT controls, lacking reverse transcriptase in the reaction, are shown. M, RNA size markers. (H) Effect of 1 μM APM for 10 d (+APM) on PLS:GUS expression in At EM101. (I) Effect of 1 μM silver nitrate for 5 d (+Ag) on PLS:GUS expression in At EM101 seedlings, leading to a spread of activity to the older part of the root. (J) Effect of 1 μM silver nitrate (+Ag) on P_PLS:GUS transgenic plants, resulting in a spread of PLS:GUS activity to the older part of the root. (K) The ethylene-overproducing eto1-1 mutation represses P_PLS:GUS expression. (L) The etr1-1 mutation leads to a spread of P_PLS:GUS expression to the older part of the root.

Figure 8.

Model for PLS Function in the Root. (A) Auxin moves into the root tip via polar auxin transport (PAT) and is also transported by this mechanism back up the root, triggering lateral root initiation. Ethylene suppresses polar auxin transport, which may prevent auxin both entering and leaving the tip. (B) Auxin is a positive regulator of PLS expression and also plays a role in cytoskeleton function, cell division, and cell expansion. Ethylene suppresses these effects, and PLS suppresses the effects of ethylene. According to this model, ethylene treatment suppresses auxin entry and exit at the root tip, and turnover of auxin would lead to a decline in the concentration of active auxin in the root tip in ethylene-treated seedlings. This in turn would lead to a reduction in PLS transcription, allowing ethylene to alter root architecture (e.g., leading to the production of thicker roots and fewer lateral roots). Auxin signaling from the shoot would antagonize this effect via PLS, which the model suggests acts as a modulator of the auxin–ethylene interaction. It is proposed that this interaction modifies root cell division, shape, and ultimately growth through effects, at least in part, on the cytoskeleton. The inhibitory effects of ethylene on auxin transport and cytoskeleton dynamics may be independent or interdependent, but they are currently unclear mechanistically.