In silico motif diversity analysis of the glycon preferentiality of plant secondary metabolic glycosyltransferases

Abstract Glycosyltransferase are the class of enzymes which specifically glycosylate various natural and artificial substrate aglycons into their glycosidic linked compounds with enhanced water solubility and transport. In several instances, glycosylation is the last step in the biosynthesis of a number of secondary plant products involving flavonoids, terpenoids, steroidal alkaloids, and saponin biosynthetic pathway. The conjugation reactions catalyzed by UGTs may therefore are critical in regulating the levels of several secondary metabolites including signaling and hormonal compounds. In this work we have analyzed genes from the databases for the presence of GT's in diverse plant families. Considerable degree of homology was seen in alignment of all available GT sequences in dicot plants as revealed by ClustalW2 and other phylogenetic tree constructing tools. Also, a highly conserved motif in their C-terminus, named the PSPG box (Plant secondary product glycosyl transferase ) was found in all the sequences through motif discovery tools e.g. MEME. The motif discovery tool identified two other distinct motifs in GT sequences, however interestingly P. patens and a putative GT sequence from A. thaliana was found to be deficient in motif 3 at N terminal of the sequence. A wide range of gene sequences were analyzed in a systematic manner to determine the structure, function and evolution of PSPG box motif found at the C-terminal

RNAi and Homologous Over-Expression Based Functional Approaches Reveal Triterpenoid Synthase Gene-Cycloartenol Synthase Is Involved in Downstream Withanolide Biosynthesis in Withania somnifera.

Withania somnifera Dunal, is one of the most commonly used medicinal plant in Ayurvedic and indigenous medicine traditionally owing to its therapeutic potential, because of major chemical constituents, withanolides. Withanolide biosynthesis requires the activities of several enzymes in vivo. Cycloartenol synthase (CAS) is an important enzyme in the withanolide biosynthetic pathway, catalyzing cyclization of 2, 3 oxidosqualene into cycloartenol. In the present study, we have cloned full-length WsCAS from Withania somnifera by homology-based PCR method. For gene function investigation, we constructed three RNAi gene-silencing constructs in backbone of RNAi vector pGSA and a full-length over-expression construct. These constructs were transformed in Agrobacterium strain GV3101 for plant transformation in W. somnifera. Molecular and metabolite analysis was performed in putative Withania transformants. The PCR and Southern blot results showed the genomic integration of these RNAi and overexpression construct(s) in Withania genome. The qRT-PCR analysis showed that the expression of WsCAS gene was considerably downregulated in stable transgenic silenced Withania lines compared with the non-transformed control and HPLC analysis showed that withanolide content was greatly reduced in silenced lines. Transgenic plants over expressing CAS gene displayed enhanced level of CAS transcript and withanolide content compared to non-transformed controls. This work is the first full proof report of functional validation of any metabolic pathway gene in W. somnifera at whole plant level as per our knowledge and it will be further useful to understand the regulatory role of different genes involved in the biosynthesis of withanolides

<i>WsCAS</i> transcript expression analysis and withanolide accumulation in Withania silencing and overexpressing lines.

<p>(A) RealTime PCR analysis of <i>WsCAS</i> silenced lines. (B) Withanolide content in silenced lines. (C) Real-Time PCR analysis of <i>WsCAS</i> overexpressing lines. (D) Withanolide content in <i>WsCAS</i> overexpressing lines.</p

Full length <i>WsCAS</i> gene with three selected silencing sites for silencing constructs preparation in <i>Withania somnifera</i>.

<p>19 bp silencing sites were enclosed in red colored boxes. Arrows indicate the region of start of amplification of particular fragment incorporating silencing sites.</p

Profiling of <i>WsCAS</i> expression and withanolide in Withania seedlings under different elicitor conditions.

<p>(A) Seedling before and after treatment of cold, wounding, SA 2mM, SA 5mM, MeJ 200μM, MeJ 500μM and heating (a-i) respectively. (B) Real time-PCR analysis of <i>WsCAS</i>. (C) Quantitative withanolide profile.</p

Withanolide biosynthetic pathway.

<p>IPP, isopentenyl pyrophosphate; MVA, mevalonate; MEP, 2-C-methyl-D-erythritol 4-phosphate GPPS, geranyl pyrophosphate synthase; FPPS, farnasyl pyrophosphate synthase; SS, squalene synthase; SE squalene epoxidase; CAS, cycloartenol synthase; LAS, lanosterol synthase; LS, lupeol synthase; BAS, beta amyrin synthase; AAS, alpha amyrin synthase.</p

Molecular characterization of Withania silenced lines and overexpressing lines of <i>WsCAS gene</i>.

<p>(A) PCR amplification with <i>bar</i> primers showing amplicon of ≈400 bp. Lane M 100 bp ladder; lane–C untransformed shoot; lane +C plasmid DNA; lane 1–9, <i>in vitro</i> transformed shoots; (B) Southern blot analysis of transgenic silenced lines of <i>Withania</i> with <i>bar</i> PCR probe. (C) PCR amplification of pIGCAS transformants with <i>npt</i>II primers showing amplicon of 500bp. Lane- 1 to 6 transformed lines. (D) Southern blot analysis of overexpresing lines.</p