Analysis of Papaya Cell Wall-Related Genes during Fruit Ripening Indicates a Central Role of Polygalacturonases during Pulp Softening

<div><p>Papaya (<i>Carica papaya</i> L.) is a climacteric fleshy fruit that undergoes dramatic changes during ripening, most noticeably a severe pulp softening. However, little is known regarding the genetics of the cell wall metabolism in papayas. The present work describes the identification and characterization of genes related to pulp softening. We used gene expression profiling to analyze the correlations and co-expression networks of cell wall-related genes, and the results suggest that papaya pulp softening is accomplished by the interactions of multiple glycoside hydrolases. The polygalacturonase <i>cpPG1</i> appeared to play a central role in the network and was further studied. The transient expression of <i>cpPG1</i> in papaya results in pulp softening and leaf necrosis in the absence of ethylene action and confirms its role in papaya fruit ripening.</p></div

Cell wall-related proteins identified in papaya pulp and their probable biological properties.

†<p>Amino acids numbers;</p>††<p>The Molecular Weight and isoelectric points were estimated using the mature proteins.</p><p>Cell wall-related proteins identified in papaya pulp and their probable biological properties.</p

Expression of cell wall-related genes during papaya ripening.

<p>Real-time PCR (qPCR) was used to analyze the mRNA levels of various genes during ripening. The column heights indicate the relative mRNA abundance; the expression values for unripened fruit one day after harvest were set to 1. The error bars on each column indicate the SD of four technical replicates from samplings I and II. The different letters represent samples that were significantly different from those collected on other days post-harvest (within the same gene), as determined by one-way ANOVA and Tukey's test (α<0.05, <i>n</i> = 4).</p

Co-expression network of papaya cell wall-related genes given by the weighted gene co-expression network analysis (WGCNA).

<p>The R package of the weighted gene co-expression network analysis (WGCNA) program was used to produce a gene association network for 25 papaya cell wall-related genes. The size of the circle indicates the weight of the gene on the network. In <b>Figures A, B, C</b> and <b>D</b>, the genes considered for the network assembly are highlighted with yellow backgrounds. <b>Figure A</b> describes the network for the papaya <i>PG</i> genes. <b>Figure B</b> describes the network for the papaya <i>GAL</i> genes. <b>Figure C</b> describes the network for the papaya <i>PME</i> genes. <b>Figure D</b> describes the network for the papaya <i>PL</i> genes. The color lines are described as positive co-expression values set to red color and negative co-expression values set to green color. The twenty-five genes are described in <b>Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105685#pone.0105685.s001" target="_blank">File S1</a></b>.</p

Transient expression of the <i>cpPG1</i> gene in papaya leaves.

<p>Constructs carrying <i>GFP</i> or <i>cpPG1-GFP</i> genes were agroinfiltrated into papaya leaves, and the GFP expression in 20-day-old leaves was observed under UV light excitation (<b>Figure A</b>). The images are representative of triplicate experiments that were performed on each agroinfiltrated leaf (<i>n</i> = 3). The absolute quantification of the <i>cpPG1</i> mRNA levels in <b>Figure B</b> demonstrate that <i>cpPG1</i> (gray bar) is transiently expressed. The light gray bar indicates data from the transient expression of <i>cpPG1</i>, the gray bars indicate data from endogenous <i>cpPG1</i> expression, and the black bars indicate the threshold cycle values (Ct) for the two genes used as internal controls (<i>cpACT</i> and <i>cp_EF1</i>). The images are representative of triplicate experiments that were performed on each agroinfiltrated leaf (<i>n</i> = 3). The figure bars are scaled to 1 cm.</p