Genome-wide identification of pistil-specific genes expressed during fruit set initiation in tomato (<i>Solanum lycopersicum</i>)

<div><p>Fruit set involves the developmental transition of an unfertilized quiescent ovary in the pistil into a fruit. While fruit set is known to involve the activation of signals (including various plant hormones) in the ovary, many biological aspects of this process remain elusive. To further expand our understanding of this process, we identified genes that are specifically expressed in tomato (<i>Solanum lycopersicum</i> L.) pistils during fruit set through comprehensive RNA-seq-based transcriptome analysis using 17 different tissues including pistils at six different developmental stages. First, we identified 532 candidate genes that are preferentially expressed in the pistil based on their tissue-specific expression profiles. Next, we compared our RNA-seq data with publically available transcriptome data, further refining the candidate genes that are specifically expressed within the pistil. As a result, 108 pistil-specific genes were identified, including several transcription factor genes that function in reproductive development. We also identified genes encoding hormone-like peptides with a secretion signal and cysteine-rich residues that are conserved among some <i>Solanaceae</i> species, suggesting that peptide hormones may function as signaling molecules during fruit set initiation. This study provides important information about pistil-specific genes, which may play specific roles in regulating pistil development in relation to fruit set.</p></div

Identification of genes preferentially expressed in pistils based on the direct-mapping method.

<p>(A) PCA analysis of the RNA-seq data. (B) Identification and validation of the expression of 532 candidate genes with RPKM values greater than 0.5 in at least one pistil sample and less than 0.5 in the other tissue samples using publically available data. Out of 532 genes, 206 were found to be expressed in the transcriptome produced by Pattison et al. (2015) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180003#pone.0180003.ref027" target="_blank">27</a>]. On the other hand, the expression of 376 genes was detected in at least one sample from many different tissues and conditions, and 275 of these showed RPKM values less than 1 in nine vegetative samples (Floral organ specific genes). Finally, by comparing the two gene sets, 108 genes were found in both sets, identified as pistil-specific genes (PSGs). (C) Heatmap of the expression of 108 genes in 17 different samples. Normalized Log2-transformed expression data were visualized by constructing a heatmap using MeV software. Hierarchical clustering by Pearson correlation was conducted. (D) Gene ontology analysis was performed using AgriGO (<a href="http://bioinfo.cau.edu.cn/agriGO/" target="_blank">http://bioinfo.cau.edu.cn/agriGO/</a>). Only one category, carboxylesterase activity (GO:0004091), was significantly (FDR<0.05) represented in the gene set, while 56 genes were not annotated and were not assigned to GO terms. bottom table; Gene ID and functional annotation described in the SGN database.</p

PSGs identified from this study.

<p>Many candidate transcriptional regulators in tomato pistils were identified, including cysteine-rich peptide (CRP)-like proteins, indicating their roles in the development of specific types of cells in the pistil. Ovule- and embryo-specific genes: <i>SlINO</i>, <i>SlTT1-like</i>, <i>SlECL1</i>, <i>SlECL5</i>. Pericarp-specific gene; <i>SlATHB13/23-like</i>. Seed coat-specific gene; <i>SlCKX8</i>.</p

List of 108 pistil-specific genes (<i>PSGs</i>) identified by the direct-mapping-based method.

<p>List of 108 pistil-specific genes (<i>PSGs</i>) identified by the direct-mapping-based method.</p

Characterization and validation of the 108 genes.

<p>(A) Many of the 108 PSGs were predominantly expressed in ovules and/or seeds in the pistil. Reads per million (RPM) values of PSGs in <i>S</i>. <i>pimpinellifolium</i> were visualized by constructing a heatmap using MeV software. Hierarchical clustering by Pearson correlation was conducted. The transcriptome data were obtained from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180003#pone.0180003.ref027" target="_blank">27</a>]. <i>OPE</i>; ovule preferentially expressed genes, <i>EPE</i>; embryo preferentially expressed genes. (B) Validation of the expression of ovule preferentially expressed (<i>OPE</i>) genes, embryo preferentially expressed (<i>EPE</i>) genes, and several transcription factor genes by RT-PCR. Most of the genes were specifically expressed in the pistil. Three pistil-specific transcription factor genes, <i>SlATHB13/23-like</i> (<i>Solyc01g010600</i>), <i>SlINO</i> (<i>Solyc05g005240</i>), and <i>SlTT1</i> (<i>Solyc10g051370</i>) showed pistil-specific expression before anthesis. Bottom one represents the expression of the internal control gene SAND [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180003#pone.0180003.ref041" target="_blank">41</a>].</p

Candidate genes for β-D-galactofuranosidase in strain JHA19.

<p>^a Based on BLAST searches using the amino acid sequences of the four ORFs, the corresponding homologs with the highest degree of identity are shown.</p><p>^b Based on CAT program predictions.</p><p>Candidate genes for β-D-galactofuranosidase in strain JHA19.</p

Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from <i>Streptomyces </i>Species

<div><p>β-D-galactofuranose (Gal<i>f</i>) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Gal<i>f</i>-ase) gene has been identified in any organism. To search for a Gal<i>f</i>-ase gene we screened soil samples and discovered a strain, identified as a <i>Streptomyces</i> species by the 16S ribosomal RNA gene analysis, that exhibits Gal<i>f</i>-ase activity for <i>4</i>-nitrophenyl β-D-galactofuranoside (<i>p</i>NP-β-D-Gal<i>f</i>) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Gal<i>f</i>-ases. Using recombinant proteins expressed in <i>Escherichia coli</i>, we found that three out of four candidates displayed the activity of not only Gal<i>f</i>-ase but also α-L-arabinofuranosidase (Ara<i>f</i>-ase), whereas the other one showed only the Gal<i>f</i>-ase activity. This novel Gal<i>f</i>-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate <i>p</i>NP-β-D-Gal<i>f</i>. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus <i>Aspergillus fumigatus</i>, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Gal<i>f</i>-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Gal<i>f</i>-specific Gal<i>f</i>-ases widely exist in microorganisms.</p></div

Relative Gal<i>f</i>-ase activity of recombinant wild-type and mutant ORF1110 proteins.

<p>^a The relative Gal<i>f</i>-ase activity was analyzed using purified recombinant proteins and <i>p</i>NP-β-D-Gal<i>f</i> as a substrate.</p><p>The relative activity of the WT protein was set as 100.</p><p>Relative Gal<i>f</i>-ase activity of recombinant wild-type and mutant ORF1110 proteins.</p

Gal<i>f</i>-ase and Ara<i>f</i>-ase activities of recombinant proteins.

<p>Gal<i>f</i>-ase and Ara<i>f</i>-ase activities were determined using <i>p</i>NP-β-D-Gal<i>f</i> (closed square, solid line) and <i>p</i>NP-α-L-Ara<i>f</i> (open triangle, dotted line) as substrates, respectively. The recombinant proteins of each ORF were used; (A), ORF0232; (B), ORF1110; (C), ORF2125; (D), ORF2812. Note that only the ORF1110 protein showed Gal<i>f</i>-ase specific activity. The activity ratios of Ara<i>f</i>-ase to Gal<i>f</i>-ase are as follows: ORF0232, 1.1:1; ORF2125, 1.8:1; ORF2812, 0.89:1.</p

Identification of the strain JHA19.

<p>Strain JHA19 (A) grown on a plate and (B) observed under microscope. (C) Phylogenetic tree of 16S rRNA gene sequences from <i>Streptomyces</i> species constructed using the neighbor-joining method. The 16S rRNA gene sequence of strain JHA19 was amplified by PCR using universal primers and genomic DNA as a template, determined by DNA sequencing and then subjected to the program CLUSTAL W for the phylogenetic analysis. The DDBJ accession numbers of the sequences used for phylogenetic comparisons are depicted.</p