Apple cluster and abscission potential.

<p>(a) central fruit (C) is clearly bigger than lateral fruits (L), and (b) schematic representation of the abscission potential of apple fruitlets inside a cluster. Lateral fruits are classified by size (L3 > L2 > L1). Darker colors represent higher abscission potentials. (adapted from Botton <i>et al</i>., 2011).</p

Gene expression at 20 DAPF.

<p>Comparison of expression of <i>MdACO1</i> (a), <i>MdPIN1</i> (b) and <i>MdAHS</i> (c) between apple central and lateral fruit seeds. Relative expressions in numbers of copies compared to the expression of the housekeeping gene encoding ubiquitin were obtained by qRT-PCR by means of the ΔΔCt method. Statistically significant differences were determined by Student’s <i>t</i>-test (<i>P</i> ≤ 0.05).</p

Dynamics of expression of apple genes.

<p>Comparison between apple central (triangles and dotted lines) and lateral fruit seeds (circles and continuous lines) of dynamics of expression of genes involved in the biosynthesis, transport or response to different hormones. (a) <i>MdPIN1</i> and <i>MdAHS</i>, involved in auxin polar transport (PAT). (b) <i>MdACO1</i>, involved in ethylene biosynthesis. (c) <i>MdETR2</i> and <i>MdETR102</i>, involved in response to ethylene. Relative expression was obtained by qRT-PCR by means of the ΔΔCt method compared to the expression of the housekeeping gene encoding ubiquitin.</p

Transcriptomic Signatures in Seeds of Apple (<i>Malus domestica</i> L. Borkh) during Fruitlet Abscission

<div><p>Abscission is the regulated process of detachment of an organ from a plant. In apple the abscission of fruits occurs during their early development to control the fruit load depending on the nutritional state of the plant. In order to control production and obtain fruits with optimal market qualities, the horticultural procedure of thinning is performed to further reduce the number of fruitlets. In this study we have conducted a transcriptomic profiling of seeds from two different types of fruitlets, according to size and position in the fruit cluster. Transcriptomic profiles of central and lateral fruit seeds were obtained by RNAseq. Comparative analysis was performed by the functional categorization of differentially expressed genes by means of Gene Ontology (GO) annotation of the apple genome. Our results revealed the overexpression of genes involved in responses to stress, hormone biosynthesis and also the response and/or transport of auxin and ethylene. A smaller set of genes, mainly related to ion transport and homeostasis, were found to be down-regulated. The transcriptome characterization described in this manuscript contributes to unravelling the molecular mechanisms and pathways involved in the physiological abscission of apple fruits and suggests a role for seeds in this process.</p></div

Summary of reads mapping.

<p>Summary of reads mapping.</p

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

<div><p>Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of <i>PIN1</i> and <i>PIN3</i> during ovule development we have used mutant alleles of both genes and also perturbed <i>PIN1</i> and <i>PIN3</i> expression using micro-RNAs controlled by the ovule specific <i>DEFH9 (DEFIFICENS Homologue 9)</i> promoter. <i>PIN1</i> down-regulation and <i>pin1-5</i> mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or <i>PIN1</i>-silenced plants. We show that sporophytically expressed <i>PIN1</i> is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.</p></div

<i>PIN1</i> and <i>PIN3</i> expression in ovules.

<p><b>A–F, </b><b><i>PIN1</i></b><b>; G–I </b><b><i>PIN3</i></b><b> A–D</b><b> </b> CLSM analysis on <i>PIN1:PIN1-GFP</i> lines.At stage 1-I PIN1 is basal polar localized in the nucellar cells shortly after the formation of the ovule primordia (<b>A</b>). At stage 2–II, PIN1 is expressed in the nucellus and in the inner integument primordia (<b>B</b>). Closer view of a nucellus (ovule stage 2–V) (<b>C</b>). In developing ovules at stage 3-II, PIN1 is still basal polar localized (<b>D</b>). (<b>E–F</b>) PIN1 immuno-localisation experiments with an anti-PIN1 antibody. PIN1 is already detectable in the ovule primordium. (<b>E</b>) ovule primordia, stage 1–I; (<b>F</b>) ovule at stage 2–I. (<b>G–I</b>) CLSM analysis of <i>PIN3:PIN3-GFP</i>. PIN3 protein is expressed in the nucellus since stage 1–II (<b>G</b>) and its expression persists until stage 3–V (<b>I</b>). PIN3 is also expressed in the funiculus vasculature throughout ovule development, in the insert a closer view of the funiculus vasculature (<b>I</b>). In (<b>J)</b> a summary of PIN1 and PIN3 localisation in developing ovules. Ovule developmental stages are labelled following the convention of Schneitz and co-workers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066148#pone.0066148-Schneitz1" target="_blank">[1]</a>. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; nu, nucellus. Scale bars: 20 µm.</p

Model of auxin flux and production during ovule development.

<p>The diagram summarises the auxin response (in green), auxin flux (red arrows) and auxin synthesis (grey) in developing ovules from stage 2–I (<b>A</b>) to stage 3–IV (<b>D</b>). At stage 2–I (<b>A</b>) the megaspore mother cell (MMC) is recognisable. At this stage the auxin response (<i>DR5rev::GFP)</i> is restricted to a few cells of the nucellus; PIN1 is also detected in the nucellar cells and has is a basal polar localization. At this stage <i>pTAA1</i> drives reporter expression in the ovule chalaza region and in the funiculus. At stage 2–III (<b>B</b>) PIN1 is detected in the inner integument primordia and in the funiculus, comcometeley with the auxin response. At stage 2–IV (<b>C</b>) the <i>TAA1</i> promoter reduces its expression in the inner integument but at the tip of the inner integument <i>YUC4</i> begins to be expressed. At this stage PIN1 and the auxin response are restricted to the funiculus since the nucellus is degenerating. The position of PIN1 in the nucellus cells strongly indicates its importance in auxin exit from these cells. The exit of auxin stimulates megagametogenesis as shown by <i>PIN1</i> silencing. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; chal, chalaza.</p

The Auxin synthesis and accumulation in wild type ovules.

<p>Wild-type <i>DR5rev:GFP</i> (<b>A–E</b>) and <i>DR5rev:3XVENUS-N7</i> (<b>F</b>) ovules were analysed at different developmental stages. The cytoplasmatic GFP signal is first detected in the nucellus of the ovule primordium from stage 1–II (<b>A</b>) until stage 3–III (<b>D</b>). <i>DR5rev:GFP</i> signal is also detected in the forming ovule vasculature (asterisks in <b>C</b> and <b>E)</b>. (<b>F</b>) the florescent signal confirms that the auxin response is only detected in the maternal nucellar cells, the YFP is fused to a nuclear localization sequence. In <b>A</b>–<b>D</b> cell membranes were stained with FM® 4–64 FX. Wild-type <i>TAA1:GFP</i> expression pattern in developing ovules at stages 2–I (<b>G</b>), 2–III (<b>H</b>), and 3–II (<b>I</b>). (<b>J</b>) YUC4:GUS is expressed since stage 3–II in the inner integument cells. The <i>YUC4</i> promoter is active in the inner integument in the cells close to the micropyle starting from stage 3–II. fg, female gametophyte; ii, inner integument; oi, outer integument; fun, funiculus; nu, nucellus Scale bars: 20 µm.</p

<i>PIN1</i> down regulation affects ovule development.

<p>(<b>A</b> and <b>B</b>) <i>pin1-5</i> ovules with defective female gametophyte. The female gametophytes arrest in development at FG1. (<b>C</b> and <b>D</b>) ovules of the PIN1 down-regulation (<i>pDEFH9:amiPIN1)</i> plants show female gametophyte defects. Female gametophytes are blocked at stages FG1 and FG3. (<b>E</b>) mature wild-type ovules, with FG7 female gametophyte. (<b>F</b>) Real-time PCR to quantify <i>PIN1</i> and <i>PIN3</i> expression levels in transgenic plants expressing the artificial microRNA against <i>PIN1</i> and <i>PIN3</i> (light grey transgenic plants showing ovule abortion, dark grey sibling wild-type plants with normal seed set). Expression of the artificial microRNA is under the control of the ovule specific <i>pDEFH9</i> promoter. Scale bars: 20 µm nu, nucellus; fun, funiculus; ii, inner integument; oi, outer integument; fg, female gametophyte.</p