Functional characterization of flavonoid 3'-hydroxylase, CsF3'H, from Crocus sativus L: Insights into substrate specificity and role in abiotic stress.

Stress-responsive dihydroxy flavonoids exhibit capability to inhibit the accretion of reactive oxygen species (ROS). The formation of these dihydroxy flavonols is catalyzed by flavonoid hydroxylases which are among the rate limiting enzymes of flavonoid biosynthesis pathway. Although flavonoid hydroxylases have been identified in several plant species but their role in abiotic stress is not explicitly documented. In the present study we report identification of all the flavonoid biosynthesis pathway genes of Crocus sativus and their expression profiling. We also report functional characterization of flavonoid 3' hydroxylase (CsF3'H) and attempt to explore its physiological role in vitro and in planta. The results indicated that CsF3'H is 1608 bp long encoding 535 amino acids. Docking and enzyme kinetic studies revealed that CsF3'H catalyzes hydroxylation of naringenin and dihydrokaempferol to eriodictoyl and dihydroquercetin respectively, but exhibits higher affinity for naringenin. Further, CsF3'H showed comparatively higher expression in floral tissues particularly stigma and its expression was significantly enhanced in response to UV-B, dehydration and salinity stress indicative of its role in stress. The expression of CsF3'H was associated with concomitant accumulation of eriodictoyl and dihydroquercetin. Transient overexpression of CsF3'H in Nicotiana benthamiana leads to the accumulation of substantial amounts of eriodictoyl and dihydroquercetin. Further, it was observed that transient expression of CsF3'H conferred tolerance to UV-B and dehydration stress as was evident from higher chlorophyll and soluble sugar and lower MDA contents. Taken together, these results suggest that CsF3'H confers tolerance to UV-B and dehydration in planta through synthesis of dihydroflavonols.

Functional Characterization of CsBGlu12, a ß-Glucosidase from Crocus sativus, Provides Insights into Its Role in Abiotic Stress through Accumulation of Antioxidant Flavonols.

Glycosylation and deglycosylation are impressive mechanisms that allow plants to regulate the biological activity of an array of secondary metabolites. Although glycosylation improves solubility and renders the metabolites suitable for transport and sequestration, deglycosylation activates them to carry out biological functions. Herein, we report the functional characterization of CsBGlu12, a ß-glucosidase from Crocus sativus. CsBGlu12 has a characteristic glucoside hydrolase 1 family (α/ß)8 triose-phosphate isomerase (TIM) barrel structure with a highly conserved active site. In vitro enzyme activity revealed that CsBGlu12 catalyzes the hydrolysis of flavonol ß-glucosides and cello-oligosaccharides. Site-directed mutagenesis of any of the two conserved catalytic glutamic acid residues (Glu200 and Glu414) of the active site completely abolishes the ß-glucosidase activity. Transcript analysis revealed that Csbglu12 is highly induced in response to UV-B, dehydration, NaCl, methyl jasmonate, and abscisic acid treatments indicating its possible role in plant stress response. Transient overexpression of CsBGlu12 leads to the accumulation of antioxidant flavonols in Nicotiana benthamiana and confers tolerance to abiotic stresses. Antioxidant assays indicated that accumulation of flavonols alleviated the accretion of reactive oxygen species during abiotic stress conditions. ß-Glucosidases are known to play a role in abiotic stresses, particularly dehydration through abscisic acid; however, their role through accumulation of reactive oxygen species (ROS) scavenging flavonols has not been established. Furthermore, only one ß-glucosidase 12 homolog has been characterized so far. Therefore, this work presents an important report on characterization of CsBGlu12 and its role in abiotic stress through ROS scavenging.

Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis.

BACKGROUND: Crocus sativus stigmas form rich source of apocarotenoids like crocin, picrocrocin and saffranal which besides imparting color, flavour and aroma to saffron spice also have tremendous pharmacological properties. Inspite of their importance, the biosynthetic pathway of Crocus apocarotenoids is not fully elucidated. Moreover, the mechanism of their stigma specific accumulation remains unknown. Therefore, deep transcriptome sequencing of Crocus stigma and rest of the flower tissue was done to identify the genes and transcriptional regulators involved in the biosynthesis of these compounds. RESULTS: Transcriptome of stigma and rest of the flower tissue was sequenced using Illumina Genome Analyzer IIx platform which generated 64,604,402 flower and 51,350,714 stigma reads. Sequences were assembled de novo using trinity resulting in 64,438 transcripts which were classified into 32,204 unigenes comprising of 9853 clusters and 22,351 singletons. A comprehensive functional annotation and gene ontology (GO) analysis was carried out. 58.5 % of the transcripts showed similarity to sequences present in public databases while rest could be specific to Crocus. 5789 transcripts showed similarity to transcription factors representing 76 families out of which Myb family was most abundant. Many genes involved in carotenoid/apocarotenoid pathway were identified for the first time in this study which includes zeta-carotene isomerase and desaturase, carotenoid isomerase and lycopene epsilon-cyclase. GO analysis showed that the predominant classes in biological process category include metabolic process followed by cellular process and primary metabolic process. KEGG mapping analysis indicated that pathways involved in ribosome, carbon and starch and sucrose metabolism were highly represented. Differential expression analysis indicated that key carotenoid/apocarotenoid pathway genes including phytoene synthase, phytoene desaturase and carotenoid cleavage dioxygenase 2 are enriched in stigma thereby providing molecular proof for stigma to be the site of apocarotenoid biosynthesis. CONCLUSIONS: This data would provide a rich source for understanding the carotenoid/apocarotenoid metabolism in Crocus. The database would also help in investigating many questions related to saffron biology including flower development.

Crocus sativus L. Tepal Extract Induces Apoptosis in Human U87 Glioblastoma Cells.

Crocus sativus (C. sativus) is considered as the costliest spice and an important medicinal plant. Herein, we investigated the effects of tepal extract (TE) of C. sativus on the viability of the human glioblastoma cells. Results revealed that TE significantly (P < 0.05) inhibited the proliferation of U87 glioblastoma cells in a dose-dependent manner with comparatively lower toxic effects against normal astrocytes. The IC50 of TE against U87 glioblastoma cells was found to be 130 µg/mL as compared to 600 µg/mL against normal astrocytes. TE also inhibited the colony formation of U87 cells significantly (P < 0.05). The AO/EB and Annexin V/PI staining assays indicated that TE stimulated apoptosis in U87 cells dose dependently. The early and late apoptotic U87 cells increased from 0.66% and 2.3% at control to 14.2% and 21.4% at 260 µg/mL of TE. Moreover, TE caused upregulation of Bax and suppression of Bcl-2. Wound healing assay showed that migration of the U87 cells was suppressed significantly (P < 0.05) at 80 µg/mL of TE. Taken together, these results suggest that TE exhibits antiproliferative effects against U87 glioma cells and may prove to be an important source of natural anticancer agents.

Overexpression of Crocus carotenoid cleavage dioxygenase, CsCCD4b, in Arabidopsis imparts tolerance to dehydration, salt and oxidative stresses by modulating ROS machinery.

Apocarotenoids modulate vital physiological and developmental processes in plants. These molecules are formed by the cleavage of carotenoids, a reaction catalyzed by a family of enzymes called carotenoid cleavage dioxygenases (CCDs). Apocarotenoids like ß-ionone and ß-cyclocitral have been reported to act as stress signal molecules during high light stress in many plant species. In Crocus sativus, these two apocarotenoids are formed by enzymatic cleavage of ß-carotene at 9, 10 and 7, 8 bonds by CsCCD4 enzymes. In the present study three isoforms of CsCCD4 were subjected to molecular modeling and docking analysis to determine their substrate specificity and all the three isoforms displayed high substrate specificity for ß-carotene. Further, expression of these three CsCCD4 isoforms investigated in response to various stresses revealed that CsCCD4a and CsCCD4b exhibit enhanced expression in response to dehydration, salt and methylviologen, providing a clue towards their role in mediating plant defense response. This was confirmed by overexpressing CsCCD4b in Arabidopsis. The transgenic plants developed longer roots and possessed higher number of lateral roots. Further, overexpression of CsCCD4b imparted enhanced tolerance to salt, dehydration and oxidative stresses as was evidenced by higher survival rate, increased relative root length and biomass in transgenic plants as compared to wild type. Transgenic plants also displayed higher activity and expression of reactive oxygen species (ROS) metabolizing enzymes. This indicates that ß-ionone and ß-cyclocitral which are enzymatic products of CsCCD4b may act as stress signals and mediate reprogramming of stress responsive genes which ultimately leads to plant defense.