Next Generation Sequencing and Transcriptome Analysis Predicts Biosynthetic Pathway of Sennosides from Senna (<i>Cassia angustifolia</i> Vahl.), a Non-Model Plant with Potent Laxative Properties

<div><p>Senna (<i>Cassia angustifolia</i> Vahl.) is a world’s natural laxative medicinal plant. Laxative properties are due to sennosides (anthraquinone glycosides) natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of <i>Cassia angustifolia</i> using Illumina MiSeq platform that resulted in a total of 6.34 Gb of raw nucleotide sequence. The sequence assembly resulted in 42230 and 37174 transcripts with an average length of 1119 bp and 1467 bp for young and mature leaf, respectively. The transcripts were annotated using NCBI BLAST with ‘green plant database (txid 33090)’, Swiss Prot, Kyoto Encylcopedia of Genes & Genomes (KEGG), Cluster of Orthologous Gene (COG) and Gene Ontology (GO). Out of the total transcripts, 40138 (95.0%) and 36349 (97.7%) from young and mature leaf, respectively, were annotated by BLASTX against green plant database of NCBI. We used InterProscan to see protein similarity at domain level, a total of 34031 (young leaf) and 32077 (mature leaf) transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to 191 KEGG pathways. There were 166 and 159 CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides. Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10,763 CDS differentially expressing in both young and mature leaf libraries of which 2,343 (21.7%) CDS were up-regulated in young compared to mature leaf. Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites. We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.</p></div

Assembly and CDS statistics of Illumina sequencing of <i>Cassia angustifolia</i> in the leaf transcriptome.

<p>Assembly and CDS statistics of Illumina sequencing of <i>Cassia angustifolia</i> in the leaf transcriptome.</p

Distribution of blast results of CDS in the C<i>assia</i>. <i>angustifolia</i> leaf transcriptome.

<p>Distribution of blast results of CDS in the C<i>assia</i>. <i>angustifolia</i> leaf transcriptome.</p

Statistics of SSRs identified using MISA in the leaf transcriptome of <i>Cassia angustifolia</i>.

<p>Statistics of SSRs identified using MISA in the leaf transcriptome of <i>Cassia angustifolia</i>.</p

GO Classification in A) young and B) mature leaf transcriptome of <i>Cassia angustifolia</i>.

<p><i>Cassia angustifolia</i> CDS were searched against the non-redundent protein sequences available in the Uni-ProtKB/SwisProt database using BLASTX with E value threshold of 1e^-06 in order to assign putative function. Out of 42,230 and 37,174 CDS in young and mature leaf respectively, 29,944 (70.9%) CDS in young and 28,099 (75.5%) CDS in mature leaf transcriptome showed significant hits to the Uni-ProtKB/SwisProt data set thereby showing overall gene conservation. In addition, many <i>C</i>. <i>angustifolia</i> transcripts showed homology to uncharacterised proteins annotated as unknown, hypothetical and expressed proteins.</p

Variation in the total sennoside content (%) with ontogeny of the leaves.

<p>Variation in the total sennoside content (%) with ontogeny of the leaves.</p

Mapping of the genes expressing differentially in young vs mature leaf on the putative sennoside biosynthetic pathway in <i>Cassia angustifolia</i>.

<p>DAHPS:3-deoxy-7-phosphoheptulonate synthase[EC:2.5.1.54], DHQS: 3-dehydroquinate synthase[EC:4.2.3.4],DHQS/SDH: 3-dehydroquinate dehydratase / shikimate dehydrogenase[EC:4.2.1.10 1.1.1.25], SMK: shikimate kinase[EC:2.7.1.71], EPSP Synthase: 3-phosphoshikimate 1-carboxyvinyltransferase/enolpyruvylshikimate phosphate synthase [EC:2.5.1.19], chorismate synthase [EC:4.2.3.5] menF: isochorismate synthase [EC:5.4.4.2], menF/menD/menH/menC or PHYLLO: isochorismate synthase / 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase / 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase / O-succinylbenzoate synthase[EC:5.4.4.2 2.2.1.9 4.2.99.20 4.2.1.113], menE: Succinylbenzoic acid-CoA ligase/acyl-activating enzyme 14 [EC:6.2.1.26], menB: naphthoate synthase[EC:4.1.3.36], DXPS: 1-deoxy-D-xylulose-5-phosphate synthase[EC:2.2.1.7], DXR: 1-deoxy-D-xylulose-5-phosphate[EC:1.1.1.267], ISPD: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase [EC:2.7.7.60], CDPMEK:4-diphosphocytidyl-2-C-methyl-D-erythritol kinase[EC:2.7.1.148], ISPF: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase[EC:4.6.1.12], HDS: (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase[EC:1.17.7.1], HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase[EC:1.17.1.2], ACCP Transferase: acetyl-CoA C-acetyltransferase [EC:2.3.1.9], HMGS:hydroxymethylglutaryl-CoA synthase [EC:2.3.3.10], HMGR: hydroxymethylglutaryl-CoA reductase (NADPH) [EC:1.1.1.34], MK: mevalonate kinase[EC:2.7.1.36], PMK: phosphomevalonate kinase [EC:2.7.4.2], MPD: diphosphomevalonate decarboxylase[EC:4.1.1.33], IPPS: isopentenyl-diphosphate delta-isomerase[EC:5.3.3.2], PKS: Polyketide Synthase, PKC:Polyketide Cyclase, UGT:UDP-Glucosyl Transferase; Y_ID:Young leaf CDS ID number for enzyme, M_ID:Mature leaf CDS ID number for enzyme, Y_FPKM: Young leaf CDS FPKM value, M_FPKM: Mature leaf CDS FPKM value, Log Fold Change: Log of change in folds of expression of CDS in young compared to matured leaf transcripts.</p

Distribution of the different classes of Transcription factors in the leaf transcriptome of <i>Cassia angustifolia</i>.

<p>Distribution of the different classes of Transcription factors in the leaf transcriptome of <i>Cassia angustifolia</i>.</p

Heat map of top 100 differentially expressed genes in young and mature leaf transcriptome of <i>Cassia angustifolia</i>.

<p>Heat map of top 100 differentially expressed genes in young and mature leaf transcriptome of <i>Cassia angustifolia</i>.</p

Clusters of orthologous groups (COG) functional classification of CDS in the young and mature leaf transcriptome of <i>Cassia angustifolia</i>.

<p>Clusters of orthologous groups (COG) functional classification of CDS in the young and mature leaf transcriptome of <i>Cassia angustifolia</i>.</p