The Molecular Mechanism of Ethylene-Mediated Root Hair Development Induced by Phosphate Starvation

<div><p>Enhanced root hair production, which increases the root surface area for nutrient uptake, is a typical adaptive response of plants to phosphate (Pi) starvation. Although previous studies have shown that ethylene plays an important role in root hair development induced by Pi starvation, the underlying molecular mechanism is not understood. In this work, we characterized an <i>Arabidopsis</i> mutant, <i>hps5</i>, that displays constitutive ethylene responses and increased sensitivity to Pi starvation due to a mutation in the ethylene receptor ERS1. <i>hps5</i> accumulates high levels of EIN3 protein, a key transcription factor involved in the ethylene signaling pathway, under both Pi sufficiency and deficiency. Pi starvation also increases the accumulation of EIN3 protein. Combined molecular, genetic, and genomic analyses identified a group of genes that affect root hair development by regulating cell wall modifications. The expression of these genes is induced by Pi starvation and is enhanced in the <i>EIN3</i>-overexpressing line. In contrast, the induction of these genes by Pi starvation is suppressed in <i>ein3</i> and <i>ein3eil1</i> mutants. EIN3 protein can directly bind to the promoter of these genes, some of which are also the immediate targets of RSL4, a key transcription factor that regulates root hair development. Based on these results, we propose that under normal growth conditions, the level of ethylene is low in root cells; a group of key transcription factors, including RSL4 and its homologs, trigger the transcription of their target genes to promote root hair development; Pi starvation increases the levels of the protein EIN3, which directly binds to the promoters of the genes targeted by RSL4 and its homologs and further increase their transcription, resulting in the enhanced production of root hairs. This model not only explains how ethylene mediates root hair responses to Pi starvation, but may provide a general mechanism for how ethylene regulates root hair development under both stress and non-stress conditions.</p></div

ChIP-qPCR assays of the binding of the EIN3 to the promoters of ten selected genes.

<p>Chromatins from the WT and <i>35S</i>::<i>EIN3-GFP</i> transgenic plants were isolated and immuno-precipitated with GFP-Trap. The levels of enrichment of the precipitated DNA fragments were quantified by qPCR assays. An actin gene fragment was amplified as the internal control. Values are means ± SD of three replicates. Experiments were repeated two times with similar results. Asterisks indicate a significant difference from the WT (<i>t</i>-test, P < 0.05).</p

Accumulation of EIN3 protein in WT, <i>hps5</i>, and ACC-treated seedlings grown under P+ and P- conditions.

<p>(A) Western blot analysis of EIN3 proteins in WT and <i>hps5</i> seedlings. (B) Western blot analysis of EIN3 proteins in WT plants treated or not treated with 10 μM ACC. In (A) and (B), the antibodies raised against native EIN3 protein were used. The proteins extracted from the seedlings of the <i>EIN3 OX</i> line were used as the positive control of EIN3 protein. (C) Confocal analysis of the accumulation of EIN3-GFP in the root tips of 7-day-old <i>35S</i>::<i>EIN3-GFP</i> seedlings grown under various conditions.</p

The genes whose expression is increased in both P+ <i>hps5</i> and P- WT.

<p>The genes whose expression is increased in both P+ <i>hps5</i> and P- WT.</p

The dominant-negative effect of the <i>hps5</i> mutation.

<p>(A), Morphology of 7-day-old seedlings of the WT, <i>hps5</i>, and three <i>35S</i>::<i>mERS1</i> transgenic lines grown on P+ and P- media. (B), A close view of the root tips of the seedlings in (A). In (A) and (B), the numbers at the top are the names of the transgenic lines.</p

Growth characteristics of WT and <i>hps5</i> mutant seedlings.

<p>(A), Morphology of 7-day-old seedlings of the WT and <i>hps5</i> grown on P+ and P- media. (B), A close view of the root tips of the WT and <i>hps5</i>.</p

Root hair phenotypes of the WT, <i>ein3</i>, <i>ein3eil1</i>, and <i>EIN3 OX</i> lines.

<p>(A), Root hair patterns of the first 3 mm of the root tips of 7-day-old WT, <i>ein3</i>, <i>ein3eil1</i>, and <i>EIN3 OX</i> seedlings grown on P+ and P- media. (B) and (C), Quantitative analyses of root hair density and length of the WT, <i>ein3</i>, <i>ein3eil1</i>, and <i>EIN3 OX</i> seedlings in (A). Values represent means ± SD of three replicates. Asterisks indicate a significant difference from the WT (<i>t</i>-test, P < 0.05).</p

Ethylene responses of the WT and various mutants.

<p>(A), Morphology of 4-day-old etiolated seedlings of the WT, <i>ein2</i>, <i>hps5</i>, and <i>ctr1</i> grown in the dark in the absence or presence of 1 μM ACC. (B), The length of the hypocotyls of the seedlings in (A). Values represent means ± SD of three replicates. (C), Relative expression as determined by qPCR of the ethylene-responsive marker genes in the 7-day-old WT and <i>hps5</i> seedlings. Values are means ± SD of three technical replications in one experiment that was performed three times with similar results. In (B) and (C), Asterisks indicate a significant difference from the WT (<i>t</i>-test, P < 0.05).</p

Molecular identification and genetic complementation of the <i>HPS5</i> gene.

<p>(A), Diagrams showing the positions of the mutations in the <i>ERS1</i> gene and ERS1 protein. Top: the structure of the <i>ERS1</i> gene. Black box: exons; grey box: 5’ and 3’ UTRs; thin line: introns; thick line: flanking sequences surrounding the <i>ERS1</i> gene. Bottom: the structure of the ERS1 protein showing the signature domains. (B), Morphology of 7-day-old seedlings and root tips of the WT, <i>hps5</i>, and complementation lines grown on P+ and P- media. (C), Quantitative analyses of root hair density and root hair length of various lines. Values represent means ± SD of three replicates. Asterisks indicate a significant difference from the WT (<i>t</i>-test, P < 0.05). For each measurement, 15–20 seedlings were used.</p

A model showing how ethylene enhances the expression of RLS4-targeted genes to promote root hair development.

<p>Above the promoter region, arrows indicate promotion, and perpendicular lines indicate inhibition.</p