Characterization of phenylalanine ammonia lyase and revealing flavonoid biosynthesis in Gymnema sylvestre R. Br through transcriptomic approach.

BACKGROUND: Gymnema sylvestre R.Br. is famous medicinal plant among diabetics for its gymnemic acid content. It also contains flavonoids, which are an essential component in various other products. Though some molecular information on the biosynthesis of gymnemic acid, polyoxypregnane, micro RNAs and photosynthetic efficiency is available, there is no gene level information available on the biosynthesis of flavonoids in this plant. RNA was extracted from winter-collected Gymnema sylvestre leaves and cDNA libraries were prepared and used for next generation sequencing. De novo transcriptome assembly were prepared and Coding DNA Sequences (CDS) of 13 major genes involved in flavonoids biosynthesis were identified from transcriptome data. Phenylalanine ammonia lyase gene containing full-length CDS was employed for in silico protein modelling and subsequent quality assessment. These models were then compared against publicly available databases. To confirm the identification of these genes, a similarity search was conducted using the NCBI BLAST tool. RESULTS: Therefore, in the present study, an effort has been made to provide molecular insights into flavonoid biosynthesis pathway by examining the expressed transcripts in G.sylvestre. Gene sequences of total thirteen major genes viz., phenylalanine ammonia lyase, 4-coumarate CoA ligase, cinnamic acid 4-hydroxylase, shikimate O-hydroxycinnamoyl transferase, coumaroyl quinate (coumaroyl shikimate) 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, naringenin 3-dioxygenase, flavanol synthase, flavonoid 3'-monooxygenase, Flavanone 7-O-glucoside 2″-O-beta-L-rhyamnosyltransferase and leucoanthocyanidin dioxygenase were identified and a putative pathway of flavonoids biosynthesis has been illustrated based on transcriptome data. CONCLUSIONS: This transcriptome study has contributed gene-level insights into the biosynthesis of flavonoids in plants as a whole and represents the first report within a non-model plant, Gymnema sylvestre perticullarly.

Altered biomass allocation and quality improvement in roots of Indian ginseng Withania somnifera Dunal. Linn. through physiological interventions.

Ashwagandha (W. somnifera Dunal. Linn.), known as Indian ginseng, contains three major bioactive compounds, withaferin-A (WA), 12-deoxywithastramonolide (WO) and withanoloide A (WD). In a field experiment, the impacts of foliar application of growth retardants/promoters was assessed with respect to biomass allocation pattern and major withanoloide content at different phenological stages in W. somnifera. Biomass accumulation pattern showed that foliar application of 500 mg l-1ethrel at 50, 65, 85, 105, and 120 days after sowing (DAS) restricted phenological progression and reduced berry weight by 61% as comparted to the control at 160 DAS. 500 mg l-1 succinic acid foliar application resulted in maximum plant height (56.4 cm), maximum dry stem weight (DWS) and dry root weight (DRW) whereas 500 mg l-1 ethrel had resulted in minimum plant height and DRW at 180 DAS. During last 50 days of crop growth, the accumulation pattern drastically changed with more than 60% of the biomass allotment to the reproductive part, the berries. The WD in roots ranged between 0.325 mg g-1and 0.342 mg g-1 during all growth stages. WA content decreased with increase in progression of crop growth and reached the lowest at 180-190 DAS. In a pot experiment, ethrel application up regulated DWF-5 by 2.44, SQE by 3.79 and CYP450s by 1.17 log2fold in roots 8 h after treatment and succinic acid had up regulated the expression of all these genes by nearly 3 log2fold change. This is in accordance with the withanoloide accumulation pattern in field condition under foliar application of these molecules.

De novo transcriptome analysis deciphered polyoxypregnane glycoside biosynthesis pathway in Gymnema sylvestre.

Gymnema sylvestre is an important medicinal plant containing antidiabetic activity. Through de novo transcriptomic study, the pathways of polyoxypregnane glycosides were explored and candidate genes of these pathways were identified in G. sylvestre. High-quality raw reads were assembled into transcripts which resulted in 193,615 unigenes. These unigenes further decoded 58,274 coding DNA sequences (CDSs). Functional annotation of predicted CDSs was carried out using the protein databases, i.e., NCBI's non-redundant, Uniprot and Pfam. Eukaryotic orthologous group (KOG) classification and transcription factor analysis has revealed most CDS-enriched categories as "Signal transduction mechanism" and "Basic Helix loop helix" (bHLH) transcription factor family, respectively. A total of 16,569 CDSs were assigned minimum one Gene Ontology (GO) term. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis disclosed 235 CDSs which represented total 27 genes of pregnane glycoside pathways and 19 CDSs represented 10 important enzymes of polyoxypregnane glycoside biosynthesis, i.e., sterol 24-C-methyltransferase, cycloeucalenol cycloisomerase, Δ14-sterol reductase, C-8,7 sterol isomerase, sterol methyltransferase 2, C-5 sterol desaturase, sterol Δ7 reductase, Δ24 sterol reductase, 3ß-hydroxysteroid dehydrogenase and progesterone 5ß reductase (5ßPOR). This transcriptome analysis provided an important resource for future functional genomic studies in G. sylvestre.

Photosynthetic characteristics of peanut genotypes under excess and deficit irrigation during summer.

In a field experiment three irrigation treatments were given to twelve peanut genotypes through drip. At 80 days after sowing (DAS) the amount of irrigation applied was 20 % higher than the evaporative demand (ET) in T1, 25 % less than ET in T2 and 48 % less than ET in T3 against the cumulative evaporative demand of 412 mm. The relative water content (RWC) of peanut leaves reduced by cutting irrigation from 93.5 % in T1 to 91.1 % in T2 and 77.2 % in T3 but, net photosynthetic rate (P N) was higher in T2 (29.6 µmol m(-2) s(-1)) than T1 (28.6 µmol m(-2) s(-1)) and T3 (24.3 µmol m(-2) s(-1)) at 75-80 DAS. Peanut genotype ICGV 91114 showed the highest P N (30.9 µmol m(-2) s(-1)) which was statistically at par with GG 20, ICGV 86590, TAG 24, SB XI, TMV 2 and TPG 41. The non-photochemical quenching (NPQ) varied with different irrigation treatment with lowest in T2 and highest in T3. The de-epoxidation state (DeS) was 38 % in T1 and T2 but, increased to 47 % in T3 due to the sever water deficit stress. Applying 20 % higher irrigation than the ET demand (T1) does not warrant any extra benefits in terms of higher photosynthesis in peanut at 75-80 DAS. Further, a reduction of 25 % of the ET (T2) in peanut seems to be the ideal condition for photosynthesis and desirable chlorophyll fluorescence parameters at 80 DAS. Girnar 3 and ICGV 91114 showed NPQ value above 2.2 and higher de-epoxidation state, maintained least deviation in Fv/Fm and Fv'/Fm' under severe water deficit condition are promising peanut genotypes.