Plant hormone receptors: new perceptions

Plant growth and development require the integration of a variety of environmental and endogenous signals that, together with the intrinsic genetic program, determine plant form. Central to this process are several growth regulators known as plant hormones or phytohormones. Despite decades of study, only recently have receptors for several of these hormones been identified, revealing novel mechanisms for perceiving chemical signals and providing plant biologists with a much clearer picture of hormonal control of growth and development

SMU-2 and SMU-1, Caenorhabditis elegans Homologs of Mammalian Spliceosome-Associated Proteins RED and fSAP57, Work Together To Affect Splice Site Choice

Mutations in the Caenorhabditis elegans gene smu-2 suppress mec-8 and unc-52 mutations. It has been proposed that MEC-8 regulates the alternative splicing of unc-52 transcripts, which encode the core protein of perlecan, a basement membrane proteoglycan. We show that mutation in smu-2 leads to enhanced accumulation of transcripts that skip exon 17, but not exon 18, of unc-52, which explains our finding that smu-2 mutations suppress the uncoordination conferred by nonsense mutations in exon 17, but not in exon 18, of unc-52. We conclude that smu-2 encodes a ubiquitously expressed nuclear protein that is 40% identical to the human RED protein, a component of purified spliceosomes. The effects of smu-2 mutation on both unc-52 pre-mRNA splicing and the suppression of mec-8 and unc-52 mutant phenotypes are indistinguishable from the effects of mutation in smu-1, a gene that encodes a protein that is 62% identical to human spliceosome-associated protein fSAP57. We provide evidence that SMU-2 protects SMU-1 from degradation in vivo. In vitro and in vivo coimmunoprecipitation experiments indicate that SMU-2 and SMU-1 bind to each other. We propose that SMU-2 and SMU-1 function together to regulate splice site choice in the pre-mRNAs of unc-52 and other genes

A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis

<div><p>The plant hormone auxin regulates numerous growth and developmental processes throughout the plant life cycle. One major function of auxin in plant growth and development is the regulation of cell expansion. Our previous studies have shown that SMALL AUXIN UP RNA (SAUR) proteins promote auxin-induced cell expansion via an acid growth mechanism. These proteins inhibit the PP2C.D family phosphatases to activate plasma membrane (PM) H⁺-ATPases and thereby promote cell expansion. However, the functions of individual PP2C.D phosphatases are poorly understood. Here, we investigated PP2C.D-mediated control of cell expansion and other aspects of plant growth and development. The nine PP2C.D family members exhibit distinct subcellular localization patterns. Our genetic findings demonstrate that the three plasma membrane-localized members, PP2C.D2, PP2C.D5, and PP2C.D6, are the major regulators of cell expansion. These phosphatases physically interact with SAUR19 and PM H⁺-ATPases, and inhibit cell expansion by dephosphorylating the penultimate threonine of PM H⁺-ATPases. <i>PP2C</i>.<i>D</i> genes are broadly expressed and are crucial for diverse plant growth and developmental processes, including apical hook development, phototropism, and organ growth. <i>GFP-SAUR19</i> overexpression suppresses the growth defects conferred by <i>PP2C</i>.<i>D5</i> overexpression, indicating that SAUR proteins antagonize the growth inhibition conferred by the plasma membrane-localized PP2C.D phosphatases. Auxin and high temperature upregulate the expression of some <i>PP2C</i>.<i>D</i> family members, which may provide an additional layer of regulation to prevent plant overgrowth. Our findings provide novel insights into auxin-induced cell expansion, and provide crucial loss-of-function genetic support for SAUR-PP2C.D regulatory modules controlling key aspects of plant growth.</p></div

<i>PP2C</i>.<i>D5</i> overexpression confers reduced cell expansion and plant growth.

<p><b>(</b>A) Shoots of 7-day-old light-grown seedlings. Scale bar = 2 mm. (B) Hypocotyl length of 7-day-old light-grown seedlings. Error bars = SD (n = 33–43). (C) Epidermal cell length of 8-day-old light-grown seedling hypocotyls. The apical most 10 cells from 10 seedlings were measured. Error bars = SD. (D) Primary root length of 7-day-old light-grown seedlings. Error bars = SD (n = 37–51). (E) Shoots of 7-day-old etiolated seedlings. Scale bar = 5 mm. (F) Hypocotyl length of 7-day-old etiolated seedlings. Error bars = SD (n = 40–49). (G) 24-day-old plants. Scale bar = 1 cm. (H) 44-day-old plants. Primary bolts were removed to better observe rosette leaves. Scale bar = 1 cm. (I) Shoots of 44 or 51-day-old plants. Scale bar = 4 cm (top) or 1 cm (bottom). (J) Flowers. Sepals and petals were removed to better observe stamen filaments. Scale bar = 1 mm. (K) Siliques. The pistils of <i>D5-HA-OX</i> lines 1 and 4 flowers were hand-pollinated with their own pollen grains. Scale bars = 2 mm. (L) 73-day-old plants. Scale bar = 4 cm. (M) Siliques. Scale bar = 4 mm. (N) Levels of Thr⁹⁴⁷-phosphorylated AHA proteins as monitored by GST-14-3-3 binding. Five micrograms of microsomal fractions prepared from 6-day-old etiolated seedlings were loaded. AHA and GST-14-3-3-bound AHA-Thr^947P proteins were detected by anti-AHA and anti-GST antibodies, respectively. (O) LiCl root inhibition assay. Six-day-old light-grown seedlings grown on ATS plates were transferred onto ATS or ATS + 10 mM LiCl plates for 3 days. New root growth after transfer was measured. Error bars = SD (n = 49–73). (P) Medium acidification assays. Eight-day-old light-grown seedlings grown on ATS plates were transferred to plates containing the pH indicator bromocresol purple (BCP, pH 6.5), and the medium color change was observed 9 (<i>D5-HA-OX</i> 1) or 12 (<i>D5-HA-OX</i> 4) days later. (B, C, D, and F) Different letters above the bars indicate significant differences (P < 0.05).</p

Differential localization of PP2C.D-GFP fusion proteins.

<p>(A) Localization of PP2C.D-GFP fusion proteins in the root tips of 5-day-old seedlings. <i>PP2C</i>.<i>D1-GFP</i> seedlings were treated with 10 μM IAA for 4 h to increase expression to detectable levels. (B) Localization of PP2C.D1-GFP fusion protein in the apical hooks of 2-day-old etiolated seedlings counter-stained with 10 μg/ml propidium iodide (PI) for 30 min. (C) Localization of PP2C.D8-GFP fusion protein in the root tips of 5-day-old seedlings. Seedlings were counter-stained with 0.5 μM MitoTracker Red CMXRos (Invitrogen) for 20 min. (A—C) Root tips and apical hooks were observed under a Nikon A1 spectral confocal microscope. Scale bars = 50 μm (A), 25 μm (B), or 10 μm (C).</p

The <i>pp2c</i>.<i>d2/5/6</i> triple mutant exhibits increased cell expansion and plant growth.

<p>(A) Eight-day-old light-grown seedlings. Scale bar = 2 mm. (B) Hypocotyl length of 8-day-old light-grown seedlings. Error bars = SD (n = 47). (C) Epidermal cell length of 8-day-old light-grown seedling hypocotyls. The apical most 10 cells from 10 seedlings were measured. Error bars = SD. (D) LiCl root inhibition assays. Five-day-old light-grown seedlings grown on ATS plates were transferred onto ATS or ATS + 10 mM LiCl plates for 3 days. New root growth after transfer was measured. Error bars = SD (n = 25–38). (E) Medium acidification assays. Six-day-old light-grown seedlings grown on ATS plates were transferred onto plates containing the pH indicator bromocresol purple (BCP, pH6.5), and the medium color change was observed 6 days later. <b>(</b>F) Levels of Thr⁹⁴⁷-phosphorylated AHA proteins as monitored by GST-14-3-3 binding. Five micrograms of microsomal fractions prepared from 6-day-old light-grown seedlings were loaded. AHA and GST-14-3-3-bound AHA proteins were detected by anti-AHA and anti-GST antibodies, respectively. (G) Shoots of 3-week-old plants. Scale bar = 1 cm. (H) Rosette leaf areas of 3-week-old plants. Total leaf areas of the plants were measured using Photoshop. Error bars = SEM (n = 23–28). (I) Flowers. Scale bars = 0.5 mm. (J) Apical hooks of 3-day-old etiolated seedlings. Scale bar = 0.5 mm. (K) Apical hook angles of 3-day-old etiolated seedlings. Error bars = SD (n = 29–35). (L) Reduced phototropic growth of <i>pp2c</i>.<i>d2/5/6</i> seedlings. Four-day-old etiolated seedlings were photo-stimulated with unilateral blue light for 2, 4, 6, and 8 h, and the angles of hypocotyl bending were measured by ImageJ. Error bars = SEM (n = 14–16). (B, C, H, and K) Different letters above the bars indicate significant differences (P < 0.05).</p

PP2C.D phosphatases interact with SAUR19 and plasma membrane H⁺-ATPases.

<p><b>(</b>A) Yeast two-hybrid assay demonstrating PP2C.D2 and SAUR19 protein interaction. Cells were plated onto appropriate selection media and grown at room temperature for 3 to 6 days. (B) Co-IP assays detecting PP2C.D5-HA and GFP-SAUR19 protein interaction. Microsomal proteins were prepared from 6-day-old etiolated seedlings. Pre-immune bleed serum (pre) or anti-GFP antibody were used for the co-IP assays. PP2C.D5-HA and GFP-SAUR19 were detected by anti-HA and anti-GFP antibodies, respectively. (C) Co-IP assays detecting PP2C.D and PM H⁺-ATPase protein interactions. Microsomal proteins were prepared from 8-day-old light-grown seedlings. Pre-immune bleed serum (pre) or anti-AHA antibody were used for the co-IP assays. PP2C.D2-GFP, PP2C.D5-GFP, PP2C.D8-GFP, and PP2C.D6-HA proteins were detected by anti-GFP and anti-HA antibodies, respectively. (D) PP2C.D expression abolishes AHA2 complementation of PM H⁺-ATPase activity in yeast. Gal, galactose; Glu, glucose; Vec, empty vector. (E). <i>In vitro</i> AHA2 dephosphorylation assays examining the dephosphorylation of AHA2-Thr^947P expressed in yeast. (B and C) 300–400 μg and 10–20 μg of microsomal proteins were used for co-IP and western blots, respectively.</p

<i>GFP-SAUR19</i> overexpression suppresses the growth defects of <i>PP2C</i>.<i>D5</i> overexpression plants.

<p>(A) Western blot analyses of GFP-SAUR19 and PP2C.D5-HA protein expression. Twenty micrograms of microsomal proteins from 8-day-old light-grown plants were loaded. GFP-SAUR19, PP2C.D5-HA, and the SEC12 loading control were detected by anti-GFP, anti-HA, and anti-SEC12 antibodies, respectively. (B) Hypocotyl length of 8-day-old light-grown seedlings. Error bars = SD (n = 33–53). (C) Primary root length of 8-day-old light-grown seedlings. Error bars = SD (n = 41–51). (D) Hypocotyl length of 7-day-old etiolated seedlings. Error bars = SD (n = 68–78). (E) 30-day-old plants. Primary bolts were removed to better observe rosette leaves. Scale bar = 1 cm. (F) Apex of 53-day-old plants. Scale bar = 1 cm. (G) Flowers. Scale bars = 2 mm. Sepals and petals were removed to better observe stamen filaments. (H) 66-day-old plants. Scale bar = 4 cm. (I) Siliques. Scale bar = 2 mm. (B-D) Different letters above the bars indicate significant differences (P < 0.05).</p

Auxin induces <i>PP2C</i>.<i>D</i> gene expression, and high temperature upregulates PP2C.D2 protein levels.

<p>(A) GUS-stained shoots of 5-day-old light-grown seedlings treated with 10 μM IAA for 4 h. Scale bars = 1 mm. (B) GUS-stained roots of 5-day-old light-grown seedlings treated with 10 μM IAA for 4 h. Scale bars = 1 mm. (C) qRT-PCR analyses of <i>SAUR</i> and <i>PP2C</i>.<i>D</i> gene expression. RNA was prepared from 3-day-old light-grown seedlings treated with 5 μM NAA or solvent control for various times. Relative expression represents expression value of NAA/expression value of mock. qRT-PCR results were based on three biological replicates. <i>S19</i>, <i>SAUR19</i>; <i>S23</i>, <i>SAUR23</i>; <i>S9</i>, <i>SAUR9</i>. Error bars = SD. (D) GUS-stained shoots of light-grown seedlings. Two or four-day-old <i>PP2C</i>.<i>D2-GUS</i> seedlings grown at 20 ^oC were shifted to 28 ^oC for 6 h, 24 h, or 5 d. GUS staining was performed at 37 ^oC for 2 h (6 h, 24 h) or 1 h (5 d). (E) Western blot analysis of PP2C.D2-GFP protein expression. Two-day-old <i>PP2C</i>.<i>D2-GFP</i> seedlings grown at 20 ^oC were shifted to 28 ^oC for 5 days. 25 micrograms of total proteins from shoots were loaded for western blot analyses using anti-GFP and anti-SEC12 antibodies. (F) qRT-PCR analyses of <i>SAUR</i> and <i>PP2C</i>.<i>D</i> gene expression. RNA was prepared from light-grown seedlings that were grown at 20 ^oC and shifted to 28 ^oC for various times. Relative expression represents expression value of 28 ^oC/expression value of 20 ^oC. qRT-PCR results were based on three biological replicates. Error bars = SD. (G) Relative hypocotyl length of 7-day-old light-grown seedlings. Relative hypocotyl length represents length value of 28 ^oC/length value of 20 ^oC. Error bars = SEM (n ≥ 22).</p

Expression patterns of <i>PP2C</i>.<i>D-GUS</i> reporters.

<p><b>(</b>A) Shoots of 5-day-old light-grown seedlings. (B) Roots of 5-day-old light-grown seedlings. (C) Shoots of 3-day-old etiolated seedlings. (D) Apical hook of a 2-day-old etiolated seedling. GUS staining was performed at 37 ^oC for 24 (A-C) or 4 h (D). Scale bars = 1 mm (A-C) or 0.5 mm (D).</p