The Relationship between Mindfulness and Readiness to Change in Alcohol Drinkers.

Mindfulness is a multi-faceted construct that involves paying attention to thoughts and emotions without automatically reacting and being critical of them. Recent research has suggested that mindfulness might play an important role in reducing problematic alcohol use. Further, Readiness to Change (RTC) is related to motivation to change drinking behaviours. The RTC scale identifies motivation to change drinking behaviours including Precontemplation, Contemplation, and Action stages. The current study investigated, for the first time, the relationship between mindfulness (and its facets) and RTC in relation to drinking behaviours. Undergraduate students from Western Sydney University (N = 279) were screened for drinking levels using the Alcohol Use Disorder Identification Test (AUDIT) and then completed the Readiness to Change Questionnaire (RCT) and the Five Facets Mindfulness Questionnaire (FFMQ), which includes the following facets: Acting with Awareness, Non-Judging of Inner Experience, Non-Reactivity to Inner Experience, Describing, and Observing. Results show that overall, mindfulness and its facets negatively correlated with RTC. Multiple regression analysis further showed that Awareness and Non-Judgement facets negatively predicted RTC. These findings provide insight into how the facets of mindfulness interact with the drinking motives of individuals and their intentions to change drinking behaviours. Based on these findings, we recommend the incorporation of mindfulness techniques in interventions targeting problematic drinking.

UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis.

Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta. Here, we analyzed UDP-glycosyltransferase (UGT) activity and diterpene levels across various developmental stages and tissues and found an apparent correlation of UGT activity with the spatiotemporal accumulation of neoandrographolide, the major diterpene C19-O-glucoside. The biochemical analysis of recombinant UGTs preferentially expressed in neoandrographolide-accumulating tissues identified a previously uncharacterized UGT86 member (ApUGT12/UGT86C11) that catalyzes C19-O-glucosylation of diterpenes with strict scaffold selectivity. ApUGT12 localized to the cytoplasm and catalyzed diterpene C19-O-glucosylation in planta. The substrate selectivity demonstrated by the recombinant ApUGT12 expressed in plant and bacterium hosts was comparable to native UGT activity. Recombinant ApUGT12 showed significantly higher catalytic efficiency using andrograpanin compared with 14-deoxy-11,12-didehydroandrographolide and trivial activity using andrographolide. Moreover, ApUGT12 silencing in plants led to a drastic reduction in neoandrographolide content and increased levels of andrograpanin. These data suggest the involvement of ApUGT12 in scaffold-selective C19-O-glucosylation of labdane diterpenes in plants. This knowledge of UGT86 function might help in developing plant chemotypes and synthesis of pharmacologically relevant diterpenes.

Application of virus-induced gene silencing in Andrographis paniculata, an economically important medicinal plant.

Kalmegh [Andrographis paniculata (Burm.f.) Wall. ex Nees] is one of the most studied medicinal plants for pharmaceutical properties and phytochemistry. However, functional genomics studies in kalmegh are so far limited due to the unavailability of a robust tool for gene silencing. Here, we tested the application of virus-induced gene silencing (VIGS) in kalmegh using the well-known Tobacco rattle virus (TRV)-based vectors and achieved targeted silencing of phytoene desaturase (ApPDS) which is essential in plants for carotenoid biosynthesis that protects chlorophyll from photooxidation. ApPDS silencing in kalmegh leaves developed a typical photobleaching phenotype. The silencing of ApPDS was confirmed by analysing ApPDS transcript level and determining chlorophyll content in the leaves of VIGS seedlings. The analysis revealed ~30% reduction in chlorophyll content, and 40 to 60% reduction in ApPDS transcript level in the leaves of VIGS seedlings. These findings clearly demonstrated the applicability of VIGS in kalmegh using TRV-based vectors. The VIGS protocol presented in this study might be useful for studying gene function related to medicinal and agricultural traits in kalmegh.

Transcriptome analysis and functional characterization of oxidosqualene cyclases of the arjuna triterpene saponin pathway.

Arjuna (Terminalia arjuna) tree has been popular in Indian traditional medicine to treat cardiovascular ailments. The tree accumulates bioactive triterpene glycosides (saponins) and aglycones (sapogenins), in a tissue-preferential manner. Oleanane triterpenes/saponins (derived from ß-amyrin) with potential cardioprotective function predominantly accumulate in the bark. However, arjuna triterpene saponin pathway enzymes remain to be identified and biochemically characterized. Here, we employed a combined transcriptomics, metabolomics and biochemical approach to functionally define a suite of oxidosqualene cyclases (OSCs) that catalyzed key reactions towards triterpene scaffold diversification. De novo assembly of 131 millions Illumina NextSeq500 sequencing reads obtained from leaf and stem bark samples led to a total of 156,650 reference transcripts. Four distinct OSCs (TaOSC1-4) with 54-71 % sequence identities were identified and functionally characterized. TaOSC1, TaOSC3 and TaOSC4 were biochemically characterized as ß-amyrin synthase, cycloartenol synthase and lupeol synthase, respectively. However, TaOSC2 was found to be a multifunctional OSC producing both α-amyrin and ß-amyrin, but showed a preference for α-amyrin product. Both TaOSC1 and TaOSC2 produced ß-amyrin, the direct precursor for oleanane triterpene/saponin biosynthesis; but, TaOSC1 transcript expressed preferentially in bark, suggesting a major role of TaOSC1 in the biosynthesis of oleanane triterpenes/saponins in bark.

Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis.

The medicinal plant sweet basil (Ocimum basilicum) accumulates bioactive ursane- and oleanane-type pentacyclic triterpenes (PCTs), ursolic acid and oleanolic acid, respectively, in a spatio-temporal manner; however, the biosynthetic enzymes and their contributions towards PCT biosynthesis remain to be elucidated. Two CYP716A subfamily cytochrome P450 monooxygenases (CYP716A252 and CYP716A253) are identified from a methyl jasmonate-responsive expression sequence tag collection and functionally characterized, employing yeast (Saccharomyces cerevisiae) expression platform and adapting virus-induced gene silencing (VIGS) in sweet basil. CYP716A252 and CYP716A253 catalyzed sequential three-step oxidation at the C-28 position of α-amyrin and ß-amyrin to produce ursolic acid and oleanolic acid, respectively. Although CYP716A253 was more efficient than CYP716A252 for amyrin C-28 oxidation in yeast, VIGS revealed essential roles for both of these CYP716As in constitutive biosynthesis of ursolic acid and oleanolic acid in sweet basil leaves. However, CYP716A253 played a major role in elicitor-induced biosynthesis of ursolic acid and oleanolic acid. Overall, the results suggest similar as well as distinct roles of CYP716A252 and CYP716A253 for the spatio-temporal biosynthesis of PCTs. CYP716A252 and CYP716A253 might be useful for the alternative and sustainable production of PCTs in microbial host, besides increasing plant metabolite content through genetic modification.

Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

Andrographis paniculata transcriptome provides molecular insights into tissue-specific accumulation of medicinal diterpenes.

BACKGROUND: Kalmegh (Andrographis paniculata) has been widely exploited in traditional medicine for the treatment of infectious diseases and health disorders. Ent-labdane-related diterpene (ent-LRD) specialized (i.e., secondary) metabolites of kalmegh such as andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide, are known for variety of pharmacological activities. However, due to the lack of genomic and transcriptomic information, underlying molecular basis of ent-LRDs biosynthesis has remained largely unknown. To identify candidate genes of the ent-LRD biosynthetic pathway, we performed comparative transcriptome analysis using leaf and root tissues that differentially accumulate ent-LRDs. RESULTS: De novo assembly of Illumina HiSeq2000 platform-generated paired-end sequencing reads resulted into 69,011 leaf and 64,244 root transcripts which were assembled into a total of 84,628 unique transcripts. Annotation of these transcripts to the Uniprot, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active Enzymes (CAZy) databases identified candidate transcripts of the ent-LRD biosynthetic pathway. These included transcripts that encode enzymes of the plastidial 2C-methyl-D-erythritol-4-phosphate pathway which provides C5 isoprenoid precursors for the ent-LRDs biosynthesis, geranylgeranyl diphosphate synthase, class II diterpene synthase (diTPS), cytochrome P450 monooxygenase and glycosyltransferase. Three class II diTPSs (ApCPS1, ApCPS2 and ApCPS3) that showed distinct tissue-specific expression profiles and are phylogenetically related to the dicotyledon ent-copalyl diphosphate synthases, are identified. ApCPS1, ApCPS2 and ApCPS3 encode for 832-, 817- and 797- amino acids proteins of 55-63 % identity, respectively. Spatio-temporal patterns of transcripts and ent-LRDs accumulation are consistent with the involvement of ApCPS1 in general (i.e., primary) metabolism for the biosynthesis of phytohormone gibberellin, ApCPS2 in leaf specialized ent-LRDs biosynthesis and ApCPS3 in root diterpene biosynthesis. Moreover, simple sequence repeats (SSRs) that might assist in genotyping and developing specific chemotypes were identified in transcripts of the specialized metabolic pathways, including ent-LRDs. CONCLUSIONS: Comparative analysis of root and leaf transcriptomes disclosed novel genes of the ent-LRD biosynthetic pathway, including three class II diTPSs that showed discrete spatio-temporal expression patterns; thus, suggesting their participation into distinct diterpene metabolic pathways of kalmegh. Overall, these results will be useful in understanding molecular basis of the medicinal ent-LRDs biosynthesis and developing breeding strategies for improving their yields.