Characterization of the Transcriptome of the Xerophyte <i>Ammopiptanthus mongolicus</i> Leaves under Drought Stress by 454 Pyrosequencing

<div><p>Background</p><p><i>Ammopiptanthus mongolicus</i> (Maxim. Ex Kom.) Cheng f., an endangered ancient legume species, endemic to the Gobi desert in north-western China. As the only evergreen broadleaf shrub in this area, <i>A</i>. <i>mongolicus</i> plays an important role in the region’s ecological-environmental stability. Despite the strong potential of <i>A</i>. <i>mongolicus</i> in providing new insights on drought tolerance, sequence information on the species in public databases remains scarce. To both learn about the role of gene expression in drought stress tolerance in <i>A</i>. <i>mongolicus</i> and to expand genomic resources for the species, transcriptome sequencing of stress-treated <i>A</i>. <i>mongolicus</i> plants was performed.</p><p>Results</p><p>Using 454 pyrosequencing technology, 8,480 and 7,474 contigs were generated after <i>de novo</i> assembly of RNA sequences from leaves of untreated and drought-treated plants, respectively. After clustering using TGICL and CAP3 programs, a combined assembly of all reads produced a total of 11,357 putative unique transcripts (PUTs). Functional annotation and classification of the transcripts were conducted by aligning the 11,357 PUTs against the public protein databases and nucleotide database (Nt). Between control and drought-treated plants, 1,620 differentially expressed genes (DEGs) were identified, of which 1,106 were up-regulated and 514 were down-regulated. The differential expression of twenty candidate genes in metabolic pathways and transcription factors families related to stress-response were confirmed by quantitative real-time PCR. Representatives of several large gene families, such as WRKY and P5CS, were identified and verified in <i>A</i>. <i>mongolicus</i> for the first time.</p><p>Conclusions</p><p>The additional transcriptome resources, gene expression profiles, functional annotations, and candidate genes provide a more comprehensive understanding of the stress response pathways in xeric-adapted plant species such as <i>A</i>. <i>mongolicus</i>.</p></div

Distribution of sequencing depth among reads.

<p>(a) Similar univariate distributions of overall depth between root and leaf of sequence reads mapped to the <i>G</i>. <i>max</i> unigenes. (b) Random distribution of depths of reads for specific unigenes compared between leaf and root sequence data. (c) Venn diagram illustrating amount of overlap in <i>G</i>. <i>max</i> unigenes to which leaf and root reads mapped at average read depth > 0.5 in both tissues.</p

Distribution of gene coverage among reads.

<p>(a) Univariate distributions of unigene sequence coverage by root vs. leaf reads mapped to the <i>G</i>. <i>max</i> unigenes. (b) Distribution of unigene sequence coverage among leaf vs. root reads with >0.1 read coverage on average, showing categorical differences in coverage between the RNA sequence sources. (c) Venn diagram showing amount of overlap in unigene sequence coverage between root and leaf tissues.</p

Species distribution of the BLAST alignment results for PUTs from <i>A</i>. <i>mongolicus</i> to the NR database.

<p>(a) Species distribution of the top BLAST alignment scores. (b) Distribution of PUTs sequence alignments lengths for matches to the NR database.</p

Differential expression analyses of the putative unique transcripts.

<p>DEGs were quantified by the RPKM method (cutoff value: FDR p-value < 0.01 and 2 fold change). The number of up- and down-regulated PUTs totalled 1106 and 514, respectively.</p

Annotation of <i>A</i>. <i>mongolicus</i> PUTs.

<p>(a) Venn diagram of the number of PUTs aligned by Blast to nr, Swiss-Prot, KEGG and COG databases. (b) COG functional classification of PUTs in <i>A</i>. <i>mongolicus</i>. A total of 11,357 PUTs were assigned to 24 classifications, as shown in the COG categories listed on the right side of histogram.</p

GO annotation of DEGs.

<p>From GO annotation results for all of the PUTs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136495#pone.0136495.s001" target="_blank">S1 Fig</a>), annotations of DEGs were summarized in the three main categories of biological process, cellular component and molecular function. The Histogram in the small insert box shows only the DEGs related to abiotic stress.</p

Size distribution of clean sequence reads and putative unique transcripts.

<p>Comparison of the length distributions of cleaned reads (a) and putative unique transcripts (b) from 454 pyrosequencing of control (CK) and drought-treated (DT) samples. Blue bars refer to CK data; Red bars refer to DT data; Green bars in graph B refer to PUTs from global (combined) assembly.</p

Distribution of transcription factors (TFs) among gene families.

<p>(a) Pie chart of percent of genes identified among TF gene families. (b) Heat map of significantly differentially expressed TFs under drought stress.</p