Chiral Chemopolymorphism in the Monoterpenes of Pistacia palaestina Leaves and Galls.

Pistacia palaestina Boiss. is a common tree in the Mediterranean maquis. The leaves of this plant accumulate defensive monoterpenes, whose levels greatly increase in galls induced by the aphid Baizongia pistaciae. We previously found a significant chemopolymorphism in monoterpene content among individual trees, but the chirality of these monoterpenes was unknown. Although most plant species specifically accumulate one enantiomeric form of a given compound, P. palaestina individuals display chemopolymorphism in the chirality of the key monoterpenes accumulated. We report here a marked enantiomeric variation for the limonene, α- and ß-pinene, camphene, sabinene, δ-3-carene, and terpene-4-ol content in leaves and galls of nine different naturally growing P. palaestina trees. Interestingly, insect-induced gall monoterpene composition is an augmentation of the specific enantiopolymorphism originally displayed by each individual tree.

Cinnamate-CoA ligase is involved in biosynthesis of benzoate-derived biphenyl phytoalexin in Malus × domestica 'Golden Delicious' cell cultures.

Apple (Malus sp.) and other genera belonging to the sub-tribe Malinae of the Rosaceae family produce unique benzoic acid-derived biphenyl phytoalexins. Cell cultures of Malus domestica cv. 'Golden Delicious' accumulate two biphenyl phytoalexins, aucuparin and noraucuparin, in response to the addition of a Venturia inaequalis elicitor (VIE). In this study, we isolated and expressed a cinnamate-CoA ligase (CNL)-encoding sequence from VIE-treated cell cultures of cv. 'Golden Delicious' (M. domestica CNL; MdCNL). MdCNL catalyses the conversion of cinnamic acid into cinnamoyl-CoA, which is subsequently converted to biphenyls. MdCNL failed to accept benzoic acid as a substrate. When scab-resistant (cv. 'Shireen') and moderately scab-susceptible (cv. 'Golden Delicious') apple cultivars were challenged with the V. inaequalis scab fungus, an increase in MdCNL transcript levels was observed in internodal regions. The increase in MdCNL transcript levels could conceivably correlate with the pattern of accumulation of biphenyls. The C-terminal signal in the MdCNL protein directed its N-terminal reporter fusion to peroxisomes in Nicotiana benthamiana leaves. Thus, this report records the cloning and characterisation of a cinnamoyl-CoA-forming enzyme from apple via a series of in vivo and in vitro studies. Defining the key step of phytoalexin formation in apple provides a biotechnological tool for engineering elite cultivars with improved resistance.

Benzoate-CoA ligase contributes to the biosynthesis of biphenyl phytoalexins in elicitor-treated pear cell cultures.

KEY MESSAGE: Benzoate-Coenzyme A ligase enzyme activity catalyzing the conversion of free benzoic acid to benzoyl-CoA was detected and biochemically characterized in the elicitor-treated pear cell cultures. Asian pear (Pyrus pyrifolia) is an economically and nutritionally important fruit-bearing tree of the subtribe Malinae. Upon pathogen attack, pears produce unique benzoate-derived biphenyl phytoalexins. The upstream biosynthesis of the biphenyl in Malinae is still incomplete. Previously, protein preparations from yeast extract-treated pear cultures were able to convert L-phenylalanine to cinnamic acid catalyzed by the activity of the phenylalanine ammonia lyase. The same extract was able to perform a C2 side-chain cleavage of cinnamic acid to benzaldehyde followed by oxidation of the latter to benzoic acid owing to the molecularly-undefined benzaldehyde synthase and benzaldehyde dehydrogenase activities, respectively. The biosynthesis of biphenyls starts with benzoate-Coenzyme A ligase (BZL), which converts benzoic acid to benzoyl-CoA. Subsequently, the previously-defined biphenyl synthase uses benzoyl-CoA to form the biphenyls. The current study reports the first time detection and characterization of BZL activity in elicitor-treated pear cell cultures. The preferred substrate was benzoic acid (Km = 62 ± 4 µM). Magnesium or manganese was prerequisite for the activity, which was enhanced by ~ 70% in the presence of potassium. Maximum BZL activity was observed 18 h post elicitation, which is in agreement with the coordinate induction reported for the enzymes in the same pathway. The induced BZL activity preceded the accumulation of biphenyls supporting its involvement in their biosynthesis.

Molecular cloning and functional analysis of a biphenyl phytoalexin-specific O-methyltransferase from apple cell suspension cultures.

MAIN CONCLUSION: This manuscript describes the cloning and functional characterization of a biphenyl phytoalexin biosynthetic gene, 3,5-dihydroxybiphenyl O-methyltransferase from elicitor-treated cell cultures of scab resistant apple cultivar 'Florina'. Apples belong to the subtribe Malinae of the Rosaceae family. Biphenyls and dibenzofurans are the specialized phytoalexins of Malinae, of which aucuparin is the most widely distributed biphenyl. The precursor of aucuparin, 3,5-dihydroxybiphenyl, is a benzoate-derived polyketide, which is formed by the sequential condensation of three molecules of malonyl-CoA and one molecule of benzoyl-CoA in a reaction catalyzed by biphenyl synthase (BIS). This 3,5-dihydroxybiphenyl then undergoes sequential 5-O-methylation, 4-hydroxylation, and finally 3-O-methylation to form aucuparin. A cDNA encoding O-methyltransferase (OMT) was isolated and functionally characterized from the cell cultures of scab-resistant apple cultivar 'Florina' (Malus domestica cultivar 'Florina'; MdOMT) after treatment with elicitor prepared from the apple scab causing fungus Venturia inaequalis. MdOMT catalyzed the regiospecific O-methylation of 3,5-dihydroxybiphenyl at the 5-position to form 3-hydroxy-5-methoxybiphenyl. The enzyme showed absolute substrate preference for 3,5-dihydroxybiphenyl. The elicitor-treated apple cell cultures showed transient increases in the MdOMT (GenBank ID MF740747) and MdBIS3 (GenBank ID JQ390523) transcript levels followed by the accumulation of biphenyls (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexins. MdOMT fused with N- and C-terminal yellow fluorescent protein showed cytoplasmic localization in the epidermis of Nicotiana benthamiana leaves. In scab inoculated greenhouse-grown 'Florina' plants, the expression of MdOMT was transiently induced in the stem followed by the accumulation of biphenyl phytoalexins.

A new enzymatic activity from elicitor-treated pear cell cultures converting trans-cinnamic acid to benzaldehyde.

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate-derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract-treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans-cinnamic acid to benzaldehyde using a non-oxidative C2 -side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C2 -side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4-hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non-elicited cell cultures. Upon yeast extract-treatment, a 13-fold increase in BS activity was observed when compared to the non-treated control cells. Moreover, feeding of the cell cultures with trans-cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans-cinnamic acid, for which the apparent Km and Vmax values were 0.5 mM and 50.7 pkat mg-1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA-independent and non-ß-oxidative route.

Role of transcriptional regulation in auxin-mediated response to abiotic stresses.

Global climate change (GCC) is posing a serious threat to organisms, particularly plants, which are sessile. Drought, salinity, and the accumulation of heavy metals alter soil composition and have detrimental effects on crops and wild plants. The hormone auxin plays a pivotal role in the response to stress conditions through the fine regulation of plant growth. Hence, rapid, tight, and coordinated regulation of its concentration is achieved by auxin modulation at multiple levels. Beyond the structural enzymes involved in auxin biosynthesis, transport, and signal transduction, transcription factors (TFs) can finely and rapidly drive auxin response in specific tissues. Auxin Response Factors (ARFs) such as the ARF4, 7, 8, 19 and many other TF families, such as WRKY and MADS, have been identified to play a role in modulating various auxin-mediated responses in recent times. Here, we review the most relevant and recent literature on TFs associated with the regulation of the biosynthetic, transport, and signalling auxin pathways and miRNA-related feedback loops in response to major abiotic stresses. Knowledge of the specific role of TFs may be of utmost importance in counteracting the effects of GCC on future agriculture and may pave the way for increased plant resilience.

Inhibitory effect of selected Indian honey on colon cancer cell growth by inducing apoptosis and targeting the ß-catenin/Wnt pathway.

Colon cancer is the most prevalent cause of death from cancer across the globe. Although chemotherapy drugs are predominantly used, their toxicity always remains a cause of concern. As an alternative to synthetic drugs, natural compounds or nutraceuticals are comparatively less toxic. Honey is widely used across different cultures as an alternative form of medicine. It represents a prominent source of plant-phenolic compounds and there is demonstrable evidence of its anti-oxidant and anti-microbial activities. The aim of the present work was to investigate the anti-proliferative effect of some Indian honeys and analyze their mechanism of action in colon cancer. In order to establish the composition-activity relationship, we evaluated the bioactive components present in selected honey samples by GC-MS and HPLC analysis. Indian honey samples showed a significant inhibitory impact on cell growth by restricting cell proliferation, causing apoptosis, and restricting the cell cycle in the G2/M phase specifically for colon cancer cells. The apoptotic activities, as imparted by the honey samples, were established by Annexin V/PI staining, real-time PCR, and immunoblot analyses. The treated cells showed increased expressions of p53 and caspases 3, 8, and 9, thus indicating the involvement of both extrinsic and intrinsic apoptotic pathways. The honey samples were also found to inhibit the ß-catenin/Wnt pathway. In the next phase of the study, the efficacy of these honey samples was evaluated in colon carcinoma induced SD-rats. Overall, these findings demonstrated that selected Indian honeys could be established as effective nutraceuticals for the prevention as well as cure of colon cancer.

Black pepper prevents anemia of inflammation by inhibiting hepcidin over-expression through BMP6-SMAD1/ IL6-STAT3 signaling pathway.

Hepcidin, a circulatory hepatic peptide hormone, is associated with systemic iron homeostasis. Inflammation leads to an increase in hepcidin expression, which dysregulates body iron level. The related disorder, anemia of inflammation, is the second most prevalent anemia-related disorder worldwide. In the present study, we conducted in vitro and in vivo studies to evaluate the effect of black pepper (BP) and its major bioactive alkaloid, piperine, on anemia of inflammation. The initial in vitro study using human hepatocyte cell line, HepG2, confirmed that among different black pepper extracts: methanol (BPME), ethanol (BPEE) and aqueous (BPAE), BPME to be most effective in downregulating transcription of hepcidin gene. Further, BPME and piperine significantly downregulated hepcidin protein expression at 200 µg/ml and 100 µM concentrations, respectively. In the next phase, BPME and piperine were found to significantly attenuate BMP-6 and IL-6 induced hepcidin overexpression by downregulating the increased level of pSMAD1 and pSTAT3 proteins, respectively. For in vivo study, we first subcutaneously injected male BALB/c mice with oil of turpentine, thrice within a period of two weeks, in order to enhance the expression of hepcidin. After that, the intraperitoneal administration of BPME and piperine at 70 and 25 mg/kg body weight, respectively, on alternate days for a period of another two weeks resulted in downregulation of hepcidin overexpression in diseased mice, as confirmed by RT-PCR and immunoblot analysis. The histopathology of liver tissue confirmed increased iron bioavailability in BPME and piperine treated animals. The molecular docking-based interaction studies demonstrated the binding potential of piperine with SMAD1 and STAT3 proteins. The binding patterns supported the proposed inhibition of hepcidin activating proteins. All together, these findings suggest black pepper as a therapeutic candidate for the treatment of anemia of inflammation.

Black pepper and piperine induce anticancer effects on leukemia cell line.

The black pepper, most commonly used in Indian cuisines for ages, is considered as "king of spices." The present study evaluates the anticancer potential of black pepper and its main constituent, i.e. alkaloid piperine, against human leukemia cell line, K-562 cells. Gas chromatography-mass spectrometry (GC-MS) analysis confirmed the presence of piperine in black pepper extract. The methanolic extract of black pepper (BP-M) and pure piperine (PIP) showed a strong cytotoxic effect against this cell line. Both BP-M and PIP generated apoptotic bodies in K-562 cells and caused nuclear condensation as visualized by fluorescent microscopy, which was further confirmed by flow cytometry analysis. BP-M and PIP also generated reactive oxygen species in K-562 cells as established by flow cytometry. The translation of Bax, caspase-3 and caspase-9 genes was found to be upregulated with subsequent downregulation of Bcl-2 gene. The anti-proliferative effect of both BP-M and PIP was also observed by trypan blue staining and was further confirmed by the downregulated expression of proliferating cell nuclear antigen (PCNA). The molecular docking studies showed the binding of PIP with PCNA and Bcl-2 and supported the in vitro findings. The docking studies also proposed the binding of PIP to ADP binding pocket of Apaf-1 protein. Taken together, these findings signify the anticancer potential of both black pepper and PIP, thus proposing black pepper as a potent nutraceutical for preventing the progression of chronic myeloid leukemia.

Salicylaldehyde synthase activity from Venturia inaequalis elicitor-treated cell culture of apple.

Salicylic acid (SA) is known to trigger a number of plant defense responses upon pathogen attack. It is well known that apple (Malus domestica) plants respond to pathogen invasion by synthesizing SA, but its biosynthesis is not well understood. In this study, we report salicylaldehyde synthase (SAS) activity from Venturia inaequalis elicitor (VIE)-treated cell suspension cultures of apple (Malus domestica 'Florina'). SAS catalyzes non-oxidative C2-side chain cleavage of 2-coumaric acid to form salicylaldehyde (SALD) in the presence of a reducing agent such as cysteine. The side chain cleavage mechanism was found to be very similar to that of salicylaldehyde synthase activity from tobacco and 4-hydroxybenzaldehyde synthase activity from Vanilla planifolia and Daucus carota. A basal SAS activity was observed in the non-elicited cell cultures, and a 7-fold increase in SAS activity was observed upon elicitation. In parallel to SAS activity, the level of total SA accumulation increased by 5.6-fold after elicitation compared to the untreated control cells. Elicitor treatment further resulted in an 8.7-fold increase in the activity of the phenylalanine ammonia-lyase (PAL) enzyme that preceded the peak of SAS activity and total SA accumulation, suggesting the involvement of the phenylpropanoid pathway in SA metabolism. The preferred substrate for SAS was 2-coumaric acid (Km = 0.35 mM), with cysteine being the preferred reducing agent. In addition, a 1.8-fold enhancement in the SA content and 0.7-fold enhancement in the SALD content was observed when elicited cell cultures were fed with 2-coumaric acid. These observations suggest the involvement of SAS in SALD biosynthesis.

Comparative metabolomics of scab-resistant and susceptible apple cell cultures in response to scab fungus elicitor treatment.

Apple scab disease caused by the fungus Venturia inaequalis is a devastating disease that seriously affects quality and yield of apples. In order to understand the mechanisms involved in scab resistance, we performed gas chromatography-mass spectrometry based metabolomics analysis of the cell culture of scab resistant cultivar 'Florina' and scab susceptible cultivar 'Vista Bella' both prior -to and -following treatment with V. inaequalis elicitor (VIE). A total 21 metabolites were identified to be altered significantly in 'Florina' cell cultures upon VIE-treatment. Among 21 metabolites, formation of three new specialized metabolites aucuparin, noraucuparin and eriobofuran were observed only in resistant cultivar 'Florina' after the elicitor treatment. The score plots of principal component analysis (PCA) exhibited clear discrimination between untreated and VIE-treated samples. The alteration in metabolite levels correlated well with the changes in the transcript levels of selected secondary metabolite biosynthesis genes. Aucuparin, noraucuparin and eriobofuran isolated from the 'Florina' cultures showed significant inhibitory effect on the conidial germination of V. inaequalis. The results expand our understanding of the metabolic basis of scab-resistance in apple and therefore are of interest in apple breeding programs to fortify scab resistance potential of commercially grown apple cultivars.

Benzaldehyde dehydrogenase-driven phytoalexin biosynthesis in elicitor-treated Pyrus pyrifolia cell cultures.

Pyrus pyrifolia (Asian pear) cell cultures respond to yeast extract (YE) treatment by accumulating benzoate-derived biphenyl phytoalexins, namely, noraucuparin and aucuparin. Biphenyl phytoalexins are defense-marker metabolites of the sub-tribe Malinae of the family Rosaceae. The substrates for biphenyl biosynthesis are benzoyl-CoA and malonyl-CoA, which combine in the presence of biphenyl synthase (BIS) to produce 3,5-dihydroxybiphneyl. In the non-ß-oxidative pathway, benzoyl-CoA is directly derived from benzoic acid in a reaction catalyzed by benzoate-CoA ligase (BZL). Although the core ß-oxidative pathway of benzoic acid biosynthesis is well-understood, the complete cascade of enzymes and genes involved in the non-ß-oxidative pathway at the molecular level is poorly understood. In this study, we report the detection of benzaldehyde dehydrogenase (BD) activity in YE-treated cell cultures of P. pyrifolia. BD catalyzes the conversion of benzaldehyde to benzoic acid. BD and BIS activities were coordinately induced by elicitor treatment, suggesting their involvement in biphenyl metabolism. Changes in phenylalanine ammonia-lyase (PAL) activity preceded the increases in BD and BIS activities. Benzaldehyde was the preferred substrate for BD (Km=52.0µM), with NAD+ being the preferred co-factor (Km=64µM). Our observations indicate the contribution of BD towards biphenyl phytoalexin biosynthesis in the Asian pear.

Development and Validation of a New HPLC Method for the Determination of Biphenyl and Dibenzofuran Phytoalexins in Rosaceae.

A simple, precise, rapid and accurate isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the simultaneous determination of biphenyl (aucuparin and noraucuparin) and dibenzofuran (eriobofuran) phytoalexin from elicitor treated cell culture of Sorbus aucuparia (mountain ash). These phytoalexins play crucial role in combating scab disease in many commercially important rosaceous plants, such as apple, pear and mountain ash. The isocratic separation was performed in a Luna C18 reversed-phase column (250 × 4.6 mm, 5 µm particle size) using a mobile phase of 1 mM trifluoroacetic acid (TFA) in water with methanol [40:60 (v/v)]. Quantization of phytoalexin was carried out on Shimadzu-HPLC system using a Photo Diode Array (PDA) detector at 254 nm by comparing the peak area. Peak purity and identity were confirmed by UV spectroscopy and ESI-MS-MS in the negative ion mode. The different analytical performance parameters such as linearity, accuracy, precision, limit of detection and limit of quantification were determined according to the International Conference on Harmonization guidelines. Linearity was observed in the concentration range of 3-400 µg/mL with excellent correlation coefficient (R(2) ≥ 0.995). This newly developed method is rapid, easy, cost-effective and can be used for monitoring scab-resistance potential of rosaceous plants.

Bilateral Florid Juvenile Fibroadenomas of the Breast in an Adolescent: A Rare Indication for Subcutaneous Mastectomy.

BACKGROUND: Juvenile fibroadenomas are rare and constitute about 0.5% to 4% of all fibroadenomas. They present as circumscribed, often large breast masses in adolescent females and are usually managed with simple enucleation. CASE: A 15-year-old girl presented with a 6-month history of rapidly growing breasts. On examination, she was found to have large, diffusely nodular breasts with marked asymmetry. Ultrasound and fine needle aspiration cytology results were suggestive of fibroadenomas, and the patient was planned for excision through a submammary incision. SUMMARY AND CONCLUSION: During surgery, both the breasts were found to be studded with nodules of varying sizes with very little normal breast tissue. A bilateral subcutaneous mastectomy was performed for complete removal of the fibroadenomas. Histopathology was reported as multiple juvenile fibroadenomas. Both breasts were later reconstructed with a latissimus dorsi myocutaneous flap with implants. We present this case to highlight a rare indication for bilateral subcutaneous mastectomy for a benign condition of the breast.