Functional Roles of Three Cutin Biosynthetic Acyltransferases in Cytokinin Responses and Skotomorphogenesis

<div><p>Cytokinins (CKs) regulate plant development and growth via a two-component signaling pathway. By forward genetic screening, we isolated an <i>Arabidopsis</i> mutant named <i>grow fast on cytokinins 1 </i> (<i>gfc1</i>), whose seedlings grew larger aerial parts on MS medium with CK. <i>gfc1</i> is allelic to a previously reported cutin mutant <i>defective in cuticular ridges</i> (<i>dcr</i>). <i>GFC1/DCR</i> encodes a soluble BAHD acyltransferase (a name based on the first four enzymes characterized in this family: Benzylalcohol O-acetyltransferase, Anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase and Deacetylvindoline 4-O-acetyltransferase) with diacylglycerol acyltransferase (DGAT) activity <i>in vitro</i> and is necessary for normal cuticle formation on epidermis <i>in vivo</i>. Here we show that <i>gfc1</i> was a CK-insensitive mutant, as revealed by its low regeneration frequency <i>in vitro</i> and resistance to CK in adventitious root formation and dark-grown hypocotyl inhibition assays. In addition, <i>gfc1</i> had de-etiolated phenotypes in darkness and was therefore defective in skotomorphogenesis. The background expression levels of most type-A <i>Arabidopsis Response Regulator</i> (<i>ARR</i>) genes were higher in the <i>gfc1</i> mutant. The <i>gfc1-</i>associated phenotypes were also observed in the cutin-deficient <i>glycerol-3-phosphate acyltransferase 4/8</i> (<i>gpat4/8</i>) double mutant [defective in glycerol-3-phosphate (G3P) acyltransferase enzymes <i>GPAT4</i> and <i>GPAT8</i>, which redundantly catalyze the acylation of G3P by hydroxyl fatty acid (OH-FA)], but not in the cutin-deficient mutant <i>cytochrome p450</i>, <i>family 86</i>, <i>subfamily A</i>, <i>polypeptide 2/aberrant induction of type three 1</i> (<i>cyp86A2/att1</i>), which affects the biosynthesis of some OH-FAs. Our results indicate that some acyltransferases associated with cutin formation are involved in CK responses and skotomorphogenesis in <i>Arabidopsis</i>.</p></div

CK levels in 1g of extracted tissue (pmol/g, Mean±SD).

<p>Asterisks indicate statistically significant differences between the mutant lines (<i>gpat4/8</i> or <i>gfc1–1</i>) and the wild type (Col-0) in an ANOVA analysis. Data are mean of four replicates (*, **, and *** correspond to <i>P</i>-values of 0.05 > <i>P</i> > 0.01, 0.01 > <i>P</i> > 0.001, and <i>P</i> < 0.001, respectively). (tZ, cZ, DHZ, iP) R: (tZ, cZ, DHZ, iP) riboside; (tZ, cZ, DHZ) OG: (tZ, cZ, DHZ) O-glucoside; (tZ, cZ, DHZ) ROG: (tZ, cZ, DHZ) riboside O-glucoside; (tZ, DHZ, iP) 7G: (tZ, DHZ, iP) 7-glucoside; (tZ, cZ, DHZ, iP) 9G: (tZ, cZ, DHZ, iP) 9-glucoside; (tZ, cZ, DHZ, iP) RMP: (tZ, cZ, DHZ, iP) riboside-5’-monophosphate; LOD: limit of detection.</p><p>CK levels in 1g of extracted tissue (pmol/g, Mean±SD).</p

<i>gfc1</i> mutants and molecular complementation.

<p>(A, B) Phenotypes of WT, <i>gfc1</i> mutant lines, and <i>pGFC1</i>:<i>GFC1 / gfc1–1</i> under long day conditions (A) or in darkness (B). (C) RT-PCR analysis (30 cycles) of the <i>GFC1</i> transcript in <i>pGFC1</i>:<i>GFC1 / gfc1–1</i>, <i>gfc1</i>, and <i>ACT-2</i> was used as a control. (D) <i>GFC1</i> gene structure and location of T-DNA insertions. p1, p2, p3 are primers for RT-PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121943#pone.0121943.s002" target="_blank">S2 Appendix</a>).</p

Comparison of CK-induced phenotypes in WT, <i>gfc1–1</i> and <i>ARR-OX</i> lines.

<p>(A) Relative fresh weight of the shoots of seedlings grown on MS with different concentrations of tZ at 7 DAG under long-day condition. (B) Relative hypocotyl length of seedlings grown on MS with different concentrations of tZ in darkness at 5 DAG. Data are mean of three biological replicates, n = 12 (A), n = 25 (B). Error bars indicate SD. Asterisks indicate statistically significant difference in the mutant lines versus the WT in a student’s t-test (*<i>P</i> <0.05 and **<i>P</i> <0.01).</p

Comparison of phenotypes of WT and various cutin-defective mutants.

<p>(A) Darkness, 14 DAG. (B) Long day conditions, 14 DAG. (C) Cuticle permeability to Toluidine blue-O.</p

Relative ethylene production.

<p>Seedlings were grown in darkness at 5 DAG. Mock-treated seedlings (WT or <i>gfc1–1</i> grown on MS) were used as controls. Relative production values = 10μM tZ (WT or <i>gfc1–1</i> grown on MS with 10μM tZ) / Mock. Data are mean of four biological replicates. Error bars indicate SD *<i>P</i> <0.05 and **<i>P</i> <0.01 (Student’s t-test) indicate significance between ‘Mock-treated’ and ‘10 μM tZ-treated’.</p

Seedlings were grown on MS with different concentrations of tZ at 7 DAG.

<p>(A) tZ-induced phenotypes. Data in (B, C) are mean of three biological replicates, n = 25 (B), 50 (C). Error bars indicate SD. Asterisks indicate statistically significant difference in the mutant lines versus the WT in a student’s t-test (*<i>P</i> <0.05 and **<i>P</i> <0.01).</p

Expression analysis of <i>GFC1</i>.

<p>Expression pattern of GUS reporter gene in <i>pGFC1</i>:<i>GUS</i> transgenic <i>Arabidopsis</i> (A-H, K). (A) 1 DAG; (B) 5 DAG; (C) Rosette leaf; (D) Inflorescence; (E) Flower; (F) Stamen; (G) Sepal and (H) Silique. (K) Seedlings were grown under long day conditions or in darkness. (I, L) Expression analysis of <i>GFC1</i> by qRT-PCR, <i>ACT-8</i> was used as an internal control. Data are means of three biological replicates, n = 3. Error bars indicate SD. (J) Expression analysis of <i>GFC1</i> by RT-PCR (30 cycles). <i>ACT-2</i> was used as a control.</p

The dark-grown phenotypes of <i>gfc1–1</i> seedlings were grown on MS with different hormones.

<p>Data in (C-F) are mean of three biological replicates with 5 DAG seedlings, n = 25. Error bars indicate SD. Asterisks indicate statistically significant differences between the mutant lines and the WT in a student’s t-test (*<i>P</i> <0.05 and **<i>P</i> <0.01).</p

Comparison of the expression levels of <i>type-A ARR</i> genes (A, C) and <i>type-B ARR</i> genes (B, D) between WT and <i>gfc1–1</i>.

<p>Seedlings were grown under long day condition (A, B) or in darkness (C, D) for 7 days. Expression levels were normalized to the <i>ACT-8</i> transcripts. WT was used as a control. Data are mean of three biological replicates, n = 3. Error bars indicate SD, *<i>P</i> <0.05 and **<i>P</i> <0.01 (Student’s t-test).</p