An in-silico approach to unravel the structure of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS): a critical enzyme for sennoside biosynthesis in Cassia angustifolia Vahl.

The laxative properties of senna are attributed to the presence of sennosides produced in the plant. The low production level of sennosides in the plant is an important impediment to their growing demand and utilization. Understanding biosynthetic pathways helps to engineer them in terms of enhanced production. The biosynthetic pathways of sennoside production in plants are not completely known yet. However, attempts to get information on genes and proteins engaged in it have been made which decode involvement of various pathways including shikimate pathway. 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) is a key enzyme involved in sennosides production through the shikimate pathway. Unfortunately, there is no information available on proteomic characterization of DAHPS enzyme of senna (caDAHPS) resulting in lack of knowledge about its role. We for the first time characterized DAHPS enzyme of senna using in-silico analysis. To the best of our knowledge this is the first attempt to identify the coding sequence of caDAHPS by cloning and sequencing. We found Gln179, Arg175, Glu462, Glu302, Lys357 and His420 amino acids in the active site of caDAHPS through molecular docking. followed by molecular dynamic simulation. The amino acid residues, Lys182, Cys136, His460, Leu304, Gly333, Glu334, Pro183, Asp492 and Arg433 at the surface interact with PEP by van der Waals bonds imparting stability to the enzyme-substrate complex. Docking results were further validated by molecular dynamics. The presented in-silico analysis of caDAHPS will generate opportunities to engineer the sennoside biosynthesis in plants.Communicated by Ramaswamy H. Sarma.

Exploring drought stress-regulated genes in senna (Cassia angustifolia Vahl.): a transcriptomic approach.

De novo assembly of reads produced by next-generation sequencing (NGS) technologies offers a rapid approach to obtain expressed gene sequences for non-model organisms. Senna (Cassia angustifolia Vahl.) is a drought-tolerant annual undershrub of Caesalpiniaceae, a subfamily of Fabaceae. There are insufficient transcriptomic and genomic data in public databases for understanding the molecular mechanism underlying the drought tolerance of senna. Therefore, the main purpose of this study was to know the transcriptome profile of senna, with special reference to drought stress. RNA from two different stages of leaf development was extracted and sequenced separately using the Illumina technology. A total of 200 million reads were generated, and a de novo assembly of processed reads in the pooled transcriptome using Trinity yielded 43,413 transcripts which were further annotated using NCBI BLAST with "green plant database (txid 33090)," Swiss Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). Out of the total transcripts, 42,280 (95.0 %) were annotated by BLASTX against the green plant database of NCBI. Senna transcriptome showed the highest similarity to Glycine max (41 %), followed by Phaseolus vulgaris (16 %), Cicer arietinum (15 %), and Medicago trancatula (5 %). The highest number of GO terms were enriched for the molecular functions category; of these "catalytic activity" (GO: 0003824) (25.10 %) and "binding activity" (GO: 0005488) (20.10 %) were most abundantly represented. We used InterProscan to see protein similarity at domain level; a total of 33,256 transcripts were annotated against the Pfam domains. The transcripts were assigned with various KEGG pathways. Coding DNA sequences (CDS) encoding various drought stress-regulated pathways such as signaling factors, protein-modifying/degrading enzymes, biosynthesis of phytohormone, phytohormone signaling, osmotically active compounds, free radical scavengers, chlorophyll metabolism, leaf cuticular wax, polyamines, and protective proteins were identified through BLASTX search. The lucine-rich repeat kinase family was the most abundantly found group of protein kinases. Orphan, bHLH, and bZIP family TFs were the most abundantly found in senna. Six genes encoding MYC2 transcription factor, 9-cis-epoxycarotenoid dioxygenase (NCED), l -ascorbate peroxidase (APX), aminocyclopropane carboxylate oxidase (ACO), abscisic acid 8'-hydroxylase (ABA), and WRKY transcription factor were confirmed through reverse transcriptase-PCR (RT-PCR) and Sanger sequencing for the first time in senna. The potential drought stress-related transcripts identified in this study provide a good start for further investigation into the drought adaptation in senna. Additionally, our transcriptome sequences are the valuable resource for accelerated genomics-assisted genetic improvement programs and facilitate manipulation of biochemical pathways for developing drought-tolerant genotypes of crop plants.

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

Senna (Cassia angustifolia 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 Cassia angustifolia 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.