Genoproteomics-assisted improvement of Andrographis paniculata: toward a promising molecular and conventional breeding platform for autogamous plants affecting the pharmaceutical industry

Andrographis paniculata (Burm. f.) Wall. ex Nees. (AP) is a hermaphroditic, self-compatible, and habitual inbreeding plant. Its main bioactive component is andrographolide, which is capable of inducing autophagic cell death in some human cancer cells and helps fight HIV/AIDS. Increasing the andrographolide content by investigating the genetic mechanisms controlling its biosynthesis in order to improve and develop high-yielding cultivars are the main breeding targets for AP. However, there might exist some limitations or barriers for crossability within AP accessions. Recently, this problem was addressed in AP by using a combination of crossbreeding and biotechnology-aided genetic methods. This review emphasizes that development of a breeding platform in a hard-to-breed plant, such as AP, requires the involvement of a broad range of methods from classical genetics to molecular breeding. To this end, a phenological stage (for example, flowering and stigma development) can be simplified to a quantitative morphological trait (for example, bud or stigma length) to be used as an index to express the highest level of receptivity in order to manage outcrossing. The outcomes of the basic crossability research can be then employed in diallel mating and crossbreeding. This review explains how genomic data could produce useful information regarding genetic distance and its influence on the crossability of AP accessions. Our review indicates that co-dominant DNA markers, such as microsatellites, are also capable of resolving the evolutionary pathway and cryptic features of plant populations and such information can be used to select the best breeding strategy. This review also highlights the importance of proteomic analysis as a breeding tool. In this regard, protein diversification, as well as the impact of normal and stress-responsive proteins on morphometric and physiological behaviors, could be used in breeding programs. These findings have immense potential for improving plant production and, therefore, can be regarded as prospective breeding platforms for medicinal plants that have an autogamous mode of reproduction. Finally, this review suggests that novel site-directed genome editing approaches such as TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) systems together with other new plant breeding technologies (NPBT) should simultaneously be taken into consideration for improvement of pharmaceutical plants

A decade of molecular understanding of withanolide biosynthesis and in vitro studies in Withania somnifera (L.) Dunal: Prospects and perspectives for pathway engineering

Withania somnifera, a multipurpose medicinal plant is a rich reservoir of pharmaceutically active triterpenoids that are steroidal lactones known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. This scenario limits biotechnological interventions for enhanced production of bioactive compounds. Nevertheless, recent emergent trends vis-à-vis, the exploration of genomic, transcriptomic, proteomic, metabolomic and in vitro studies have opened new vistas regarding pathway engineering of withanolide production. During recent years, various strategic pathway genes have been characterized with significant amount of regulatory studies which allude towards development of molecular circuitries for production of key intermediates or end products in heterologous hosts. Another pivotal aspect covering redirection of metabolic flux for channelizing the precursor pool towards enhanced withanolide production has also been attained by deciphering decisive branch point(s) as robust targets for pathway modulation. With these perspectives, the current review provides a detailed overview of various studies undertaken by the authors and collated literature related to molecular and in vitro approaches employed in W. Somnifera for understanding various molecular network interactions in entirety

Dynamics of essential oil biosynthesis in relation to inflorescence and glandular ontogeny inSalvia sclarea

Changes in the essential oil concentration, composition and glandular morphology of Salvia sclarea L. were studied at different stages of inflorescence maturity. The chemical composition of oil was determined by GC–MS, NMR and the peak enrichment technique. The oil yield at bud stage on a fresh basis was minimum (0.08%), peaked at full bloom stage (0.18%) and was followed by sharp decline on maturation (0.07%). The main components of the oil were linalool (36.6–41.9%) and linalyl acetate (13.2–19.2%). The maximum percentage composition of various constituents was coincident with full bloom stage. β -Humulene (6.4–8.9%), α -cadinene (t–1.5%), β -caryophyllene (t–1.4%), β - caryophyllene oxide (0.4–1.4%) and sclareol (0.3–1.8%) present in the oil showed gradual increase in percentage over the different stages of maturity, with no significant turnover losses at maturation stage. Turnover of essential oil (61.1% loss) and monoterpenes (23.6% loss) occurred late in development at full maturity. Scanning electron microscopy (SEM) was used to follow the changes in the oil secretory glands over different temporal phases of maturation. The decline in oil concentration and monoterpene constituents compared very well with the observed deterioration and lyses of secretory glandular system. An abrupt fall in oil concentration apparently appears due to differential evaporation of the more volatile constituents, rather than as a dynamic balance between biosynthetic and catabolic processes

Comparative analysis of genetic diversity using molecular and morphometric markers in Andrographis paniculata (Burm. f.) Nees

Andrographis paniculata is a medicinal plant of immense therapeutic value. The present study was aimed to elucidate its genetic diversity based on morphochemical and RAPD markers from 53 accessions belonging to 5 ecogeographic regions. Analysis of variance and D2 statistics revealed significant differences in all the metric traits and sufficient inter-cluster distances indicating considerable diversity among the accessions. The complementary approach of RAPD was used to evaluate the genetic dissimilarities among all the accessions using 6 highly polymorphic primers. The average proportion of polymorphic loci across primers was 96.28%. The molecular genetic diversity based on Shannon index per primer averaged 5.585 with values ranging from 3.08 to 8.70 indicating towards wide genetic base. RAPD based UPGMA and D2 cluster analysis also revealed that various accessions available in different eco-geographic regions might have originated from native places of wild abundance. Similarity matrices were generated for molecular markers and morphometric data to determine the degree of congruence between the two. A highly significant but low correlation(r = 0.547, P < 0.001) was obtained thus implying the correspondence between the two. The species is hermaphroditic and a habitual inbreeder. The present study yielded a typical triangular congruence between its breeding system, morphometric traits and RAPD markers thus elucidating the usefulness of complementary approaches to make diversity analysis more explanatory and purposeful for optimum genetic amelioration and effective conservation of its genotypic variability

Kinetic study of <i>Ga</i>CHSs using different substrates.

<p>The kinetic parameters K_m and V_max were calculated by nonlinear regression analysis in order to determine the relative efficiency of <i>Ga</i>CHS1 and <i>Ga</i>CHS2 against the different substrates including p-coumaroyl-CoA, Acetyl-CoA, Butyryl-CoA, Hexanoyl-CoA and Octanoyl CoA.</p

Predicted three-dimensional models and ligand-binding sites of <i>Ga</i>CHSs.

<p>Ribbon model display of the three-dimensional structures of <i>Ga</i>CHS1 and <i>Ga</i>CHS2 with conserved catalytic triad (Cys-His-Asn) shown in the central core of the structures <b>(a and d)</b>;Ribbon model display of the three-dimensional structures of <i>Ga</i>CHS1 <b>(b)</b> and <i>Ga</i>CHS2 <b>(e)</b> as predicted by I-TASSER web server, showing geometry of active site a malonyl-CoA binding motif shown as mesh structures and gatekeepers Phe²¹⁵and Phe²⁶⁵ (Pink in <i>Ga</i>CHS1 and Red in <i>Ga</i>CHS2). The ligand binding sites as predicted by 3DLigandSite web server are depicted in the ribbon model <b>(c and f)</b>. The predicted ligand binding sites for <i>Ga</i>CHS1 are Ala²¹¹, Gln²¹², Ala²¹³, Leu²¹⁴, Phe²¹⁵, Ile²⁵⁴, Phe²⁶⁵, Leu²⁶⁷, Lys²⁶⁹, Val²⁷¹, Pro²⁷², Gly³⁰⁵, Gly³⁰⁶, Ala³⁰⁸, Ile³⁰⁹, Ile³³⁶ and for <i>Ga</i>CHS2, the predicted ligand binding sites were Lys⁵⁵, Phe⁵⁶, Asp⁵⁷, Leu⁵⁸, Ser⁵⁹, Ala⁶⁰, Val⁶², Thr⁶³, Ile⁶⁴, Leu¹⁶⁴, Leu²⁰⁶, Asp²⁰⁷, Leu²⁰⁹, Val²¹⁰, Gly²¹¹, Leu²¹⁴, Phe²¹⁵, Ile²⁵⁴, Phe²⁶⁵, Leu²⁶⁷, Lys²⁶⁹, Val²⁷¹, Pro²⁷², Gly³⁰⁵, Gly³⁰⁶, Ala³⁰⁸, Asn³³⁶.</p

Phylogenetic analysis of <i>Ga</i>CHS-1 and <i>Ga</i>CHS-2.

<p>The phylogenetic analysis was performed based on Maximum Likelihood methodwith 1000 bootstrap replicates using the MUSCLE program and MEGA- 5 software. The analysis involved alignment of 34 amino acid sequences which were chosen by BLAST search of <i>Ga</i>CHS genes from NCBI data-base. The desired sequences were selected based on the complete cds information available. The evolutionary distances were computed using the Poisson correction method. The database accession numbers of the CHS sequences used are as follows: <i>Grewiaasiatica</i>CHS-1(KX129910), <i>Grewia asiatica</i>CHS-2 (KX129911), <i>Gossypiumhirsutum</i>(AEO96985.1), <i>Gossypiumarboreum</i> CHS-1 (KHG25969.1), <i>Gossypiumraimondii</i>(XP_012454899.1), <i>Theobroma cacao</i> (EOY05368.1), <i>Abelmoschusesculentus</i>(AGW22222.1), <i>Abelmoschusmanihot</i>(ACE60221.1), <i>Gossypiumhirsutum CHS2</i> (AEO96988.1), <i>Gossypiumraimondii</i>CHS1 (XP_012455000.1), <i>Gossypiumarboreum</i>(KHG14899.1), <i>Gossypiumhirsutum</i>CHS1 (ACV72638.1), <i>Gossypiumraimondii</i>CHS2 (XP_012440802.1), <i>Hibiscus cannabinus</i>CHS1 (AIC75908.1), <i>Hibiscus cannabinus</i>CHS2 (AIA22214.1), <i>Mangiferaindica</i>CHS1 (AIY24986.1), <i>Mangiferaindica</i>CHS (AIB06736.1), <i>Mangiferaindica</i>CHS (AIY24987.1), <i>Camellia sinensis</i>(AGI02994.1), <i>Camellia japonica</i> (BAI66465.1), <i>Ziziphus jujube</i> (XP_015887549.1), <i>Populustrichocarpa</i>(EEE78799.1), <i>Pyruscommunis</i> (AAX16494.1), <i>Malus hybridcultivar</i>(ACN25139.1), <i>Malus domestica</i>CHS2 (AFX71920.1), <i>Malus domestica</i>CHS1 (AGE84303.1), <i>Malus domestica</i>PREDICTED:polyketidesynthase5-like (XP_008380608.1), <i>Medicago sativa</i> CHS(AAB41559.1), <i>Silenelittorea</i> CHS1 (AMQ23617.1), <i>Nicotiana tabacum</i> CHS (AAK49457.1), <i>Gypsophila paniculata</i>CHS(AAP74755.1), <i>Polygonum cuspidatum</i> CHS (AFD64563.1), <i>Fagopyrumtataricum</i>CHS(ADG02377.1), <i>Arabidopsis</i>CHS(AAB35812.1). The bar indicates an evolutionary distance of 0.02%.</p

Multiple sequence alignment of deduced amino acid sequencesof <i>Ga</i>CHS-1 and <i>Ga</i>CHS-2 with related plant CHS sequences using Clustal Omega multiple sequence alignment tool.

<p>Functionally important conserved residues are highlighted with a coloured background: Dark yellow, the four catalytic residues (Cys-His-Asp triad + F) that are conserved in all chalcone synthases; red, the 13 residues that shape the geometry of the active site; pink, the malonyl-CoA binding motif; and green, the highly conserved CHS signature sequence, N-myristoylation motif. (GenBank accession numbers): <i>Grewiaasiatica</i>CHS-1(KX129910), <i>Grewiaasiatica</i>CHS-2 (KX129911),<i>Hibiscus cannabinus</i>CHS1 (AIC75908.1), <i>Bv</i>CHS, <i>Theobroma cacao</i> (EOY05368.1), <i>Gossypiumraimondii</i>(XP_012454899.1), <i>Gossypiumarboreum</i>(KHG14899.1), <i>Abelmoschusesculentus</i>(AGW22222.1), <i>Abelmoschusmanihot</i>(ACE60221.1).</p

Molecular and functional characterization of two isoforms of chalcone synthase and their expression analysis in relation to flavonoid constituents in <i>Grewia asiatica</i> L

<div><p>Chalcone synthase constitutes a functionally diverse gene family producing wide range of flavonoids by catalyzing the initial step of the phenylpropanoid pathway. There is a pivotal role of flavonoids in pollen function as they are imperative for pollen maturation and pollen tube growth during sexual reproduction in flowering plants. Here we focused on medicinally important fruit-bearing shrub <i>Grewia asiatica</i>. It is a rich repository of flavonoids. The fruits are highly acclaimed for various putative health benefits. Despite its importance, full commercial exploitation is hampered due to two drawbacks which include short shelf life of its fruits and larger seed volume. To circumvent these constraints, seed abortion is one of the viable options. Molecular interventions tested in a number of economic crops have been to impair male reproductive function by disrupting the chalcone synthase (CHS) gene activity. Against this backdrop the aim of the present study included cloning and characterization of two full-length cDNA clones of <i>Ga</i>CHS isoforms from the CHS multigene family. These included <i>Ga</i>CHS1 (NCBI acc. KX129910) and <i>Ga</i>CHS2 (NCBI acc. KX129911) with an ORF of 1176 and 1170 bp, respectively. <i>Ga</i>CHSs were heterologously expressed and purified in <i>E</i>. <i>coli</i> to validate their functionality. Functionality of CHS isoforms was also characterized via enzyme kinetic studies using five different substrates. We observed differential substrate specificities in terms of their K_m and V_max values. Accumulation of flavonoid constituents naringenin and quercetin were also quantified and their relative concentrations corroborated well with the expression levels of <i>Ga</i>CHSs. Further, our results demonstrate that <i>Ga</i>CHS isoforms show differential expression patterns at different reproductive phenological stages. Transcript levels of <i>Ga</i>CHS2 were more than its isoform <i>Ga</i>CHS1 at the anthesis stage of flower development pointing towards its probable role in male reproductive maturity.</p></div