Identification and in vivo functional analysis of furanocoumarin-responsive cytochrome P450s in a Rutaceae-feeding Papilio butterfly.

The Order Lepidoptera contains nearly 160,000 described species and most of them are specialist herbivores that use restricted plant species as hosts. Speciation that originated from host shift is one of the important factors for the diversification of Lepidoptera. Because plants prepare secondary metabolites for defense against herbivores, with varying profiles of the components among different plant taxa, the specialist herbivores need to be adapted to the toxic substances unique to their host plants. Swallowtail butterflies of the genus Papilio consist of over 200 species. Approximately 80% of them utilize Rutaceae plants, and among the remaining species, a specific subgroup uses phylogenetically distant Apiaceae plants as larval hosts. Rutaceae and Apiaceae commonly contain toxic secondary metabolites, furanocoumarins, and molecular phylogenetic studies support the concept that Apiaceae feeders were derived from Rutaceae feeders. Molecular mechanisms underlying furanocoumarin tolerance in Papilio butterflies have been investigated almost exclusively in an Apiaceae feeder by an in vitro assay. In contrast, there is little information regarding the Rutaceae feeders. Here, we focused on a Rutaceae feeder, Papilio xuthus, and identified two furanocoumarin-responsive cytochrome P450-6B (CYP6B) genes, of which one was an ortholog of a furanocoumarin-metabolizing enzyme identified in the Apiaceae-feeding Papilio while the other was previously unreported. We further conducted in vivo functional analysis using the CRISPR/Cas9 system, revealing a contribution of these CYP6Bs to furanocoumarin tolerance of P. xuthus larvae. Our findings suggest that co-option of furanocoumarin-metabolizing CYP6B enzymes at least partially contributed to the host shift from Rutaceae to Apiaceae in Papilio butterflies.

Novel studies on Isolation, purification and characterization of dibenzonitro compound from Glycosmis pentaphylla (Retz.) DC. and effect in downregulating neuronal cancers.

To isolate Letrozole from Glycosmis pentaphylla (Retz.) DC. and to determine its effect on regulating the proliferation, cell cycle distribution, apoptosis and key mechanisms in human neuroblastoma cell lines. Letrozole was isolated through column chromatographic technique and its effect was checked on human neuroblastoma cell lines, IMR 32. The effects of Letrozole on cell viability were measured by MTT assay, and the cell cycle distribution was determined by flow cytometry. The expression changes in mRNA of proliferating cell nuclear antigen (PCNA), cyclin D1 and Bcl-xL were taken from real-time PCR analysis and the protein levels were detected by Western blotting. The results of the present study showed that Letrozole, isolated from leaves of G. pentaphylla could cause significant inhibitory effect on proliferation of IMR 32 cells in a dose dependent manner. Cell arrest was obtained at S phase with the treatment of Letrozole. Apart from this, the expression of PCNA, cyclin D1 and Bcl-xL were decreased both at mRNA and protein levels for the same treatment. Letrozole can inhibit proliferation, induce cell arrest and cause apoptosis in IMR 32 cell lines. The decreased expression of PCNA, cyclin D1 and Bcl-xL induced by Letrozole contributes to the above effects in vitro. This is the first report on the isolation of Letrozole from G. pentaphylla.

Variation in microbial feature perception in the Rutaceae family with immune receptor conservation in citrus.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in 2 subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22), and chitin, including 1 feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized 2 genetically linked FLS2 homologs from "Frost Lisbon" lemon (Citrus ×limon, responsive) and "Washington navel" orange (Citrus ×aurantium, nonresponsive). Surprisingly, FLS2 homologs from responsive and nonresponsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. "Washington navel" orange weakly responded to chitin, whereas "Tango" mandarin (C. ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the 2 genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Chloroxylon swietenia (Roxb.) DC induces cell death and apoptosis by down-regulating the NF-κB pathway in MCF-7 breast cancer cells: In vitro and in vivo investigations.

BACKGROUND: Natural products with targeted bioactivity have gained major attention in the field of cancer research owing to emerging anti-cancer drug resistance and off target toxicities. Chloroxylon swietenia (Roxb.) DC is recognized as a folklore medicinal plant and has numerous therapeutic benefits in the folklore medicine system, however the anti-cancer potential of this plant and its mechanism of action is poorly understood. AIMS: The aim of the study was to investigate the anti-breast cancer efficacy of C. swietenia leaves methanol extract (CSLME) against MCF-7 hormone dependent human breast cancer cell line with possible mechanism of action. METHODS AND RESULTS: The anti-breast cancer activity of CSLME against MCF-7 cells was assessed by evaluating its efficacy toward cytotoxicity, cell migration, colony formation, DNA fragmentation, apoptosis, cytoskeleton, angiogenesis, cell cycle regulation, and animal toxicity. The preliminary screening of CSLME against MCF-7 cells revealed the cytotoxicity (IC50 20 µg/ml), inhibited cell migration, colony formation, and angiogenesis. It was observed that CSLME induces apoptosis by nuclear fragmentation and disruption of cytoskeleton by actin derangement. The results of Annexin V-FITC assay and cell cycle analysis by flow cytometry clearly pointed out the sizable fraction of apoptotic cells, and arrested the cells at G2/M phase of cell cycle. The results of the immunoblotting experiments showed that CSLME activates intrinsic pathway of apoptosis with down regulation of anti-apoptotic marker like Bcl2, up regulation of pro-apoptotic markers like Bax & Bad, along with successful cleavage of Caspase-9 and PARP-1. Further, western blot analysis revealed the possible down regulation of NF-κB pathway by CSLME, which may be responsible for anti-cancer activity in MCF-7 cells. In vivo animal model studies using NOD-SCID mice demonstrated impressive anti-tumor activity with significant reduction in tumor volume of MCF-7 tumor xenograft. Of note, in-vivo acute oral toxicity study as per Organization for Economic Cooperation and Development 423 revealed the nontoxic nature of CSLME. CONCLUSION: The in vitro and in vivo findings clearly outline the potential of CSLME as inhibitor of growth and proliferation of MCF-7 cells. Mechanistically, CSLME seems to activate intrinsic pathway of apoptosis, arrest cell cycle, target actin cytoskeleton, inhibit growth, colony formation, migration, and angiogenesis, with down regulation of NF-κB pathway leading to cell death.

Two Dof transcription factors promote flavonoid synthesis in kumquat fruit by activating C-glucosyltransferase.

Although DNA binding with one finger (Dof) constitutes a crucial plant-specific family of transcription factors (TFs) that plays important roles in a wide range of biological processes, the molecular mechanisms underlying Dof regulation of flavonoid biosynthesis in plants remain largely unknown. Here, we characterized 28 Dof genes (FhDof1-FhDof28) from the 'Hongkong' kumquat (Fortunella hindsii) cultivar genome. Promoter analysis and transcriptome profiling revealed that four FhDofs - FhDof4, FhDof9, FhDof15, and FhDof16 - may be involved in flavonoid biosynthesis through binding to the flavonoid C-glycosyltransferase (FhCGT) promoter. We cloned homologous genes of four FhDofs, designated as FcDof4, FcDof9, FcDof15, FcDof16, and a homologous gene of FhCGT, designated as FcCGT, from the widely cultivated 'HuaPi' kumquat (F. crassifolia). Quantitative reverse transcription-polymerase chain reaction analysis revealed that FcDof4 and FcDof16 were significantly correlated with FcCGT expression during development stages in the 'HuaPi' fruit (Pearson's correlation coefficient > 0.7) and were localized to the nucleus. Results of yeast one-hybrid, electrophoretic mobility shift, and dual-luciferase assays indicated that the two FcDofs trigger FcCGT expression by specifically binding to its promoters. Moreover, transient overexpression of FcDof4 and FcDof16 enhances the transcription of structural genes in the flavonoid biosynthetic pathway and increases C-glycosylflavonoid content. Our results provide strong evidence that the TFs FcDof4 and FcDof16 promote flavonoid synthesis in kumquat fruit by activating FcCGT expression.

Oxyprenylated Secondary Metabolites as Modulators of Lipid and Sugar Metabolism.

O-Prenylcoumarins (3,3-dimethylallyl, geranyl-, farnesyl- and related biosynthetic derivatives) represent a class of rarely occurring natural compounds. Most of these secondary metabolites have been obtained from plant species belonging to the Rutaceae, Apiaceae, and Fabaceae families, and from fungi, and bacteria. In the last two decades, prenyloxycoumarins have been found to possess great potential in terms of pharmacological activities. The aim of this comprehensive review is to make a survey of the so far reported literature citations about these valuable phytochemicals and structurally related compounds about their modulatory properties of lipid and sugar metabolism. Literature data have been acquired from the main Internet database. Several oxyprenylated secondary metabolites have been surveyed. Among these, prenyloxycoumarins represented the main group displaying valuable effects as modulators of lipid and sugar metabolism. The title phytochemicals have been found in common fruits and vegetables already known to have beneficial effects, thus enforcing the nutraceutical role of these food plants. All compounds outlined in the present review article have a great potential in future for the prevention and management of acute and chronic metabolic disorders.

Identification of genes associated with soluble sugar and organic acid accumulation in 'Huapi' kumquat (Fortunella crassifolia Swingle) via transcriptome analysis.

BACKGROUND: The levels and ratios of sugar and acid are important contributors to fruit taste. Kumquat is one of the most economically important citrus crops, but information on the soluble sugar and organic acid metabolism in kumquat is limited. Here, two kumquat varieties - 'Rongan' (RA) and its mutant 'Huapi' (HP) - were used to assess soluble sugar and organic acid accumulation and the related genes. RESULTS: Soluble sugars include sucrose, glucose and fructose, while malate, quinic acid and citrate are the dominant organic acids in the fruits of both kumquat varieties. HP accumulated more sugars but fewer organic acids than did RA. Transcriptome analysis revealed 63 and 40 differentially expressed genes involved in soluble sugar and organic acid accumulation, respectively. The genes associated with sugar synthesis and transport, including SUS, SPS, TST, STP and ERD6L, were up-regulated, whereas INVs, FRK and HXK genes related to sugar degradation were down-regulated in HP kumquat. For organic acids, the up-regulation of PEPC and NAD-MDH could accelerate malate accumulation. In contrast, high expression of NAD-IDH and GS resulted in citric acid degradation during HP fruit development. Additionally, the PK, PDH, PEPCK and FBPase genes responsible for the interconversion of soluble sugars and organic acids were also significantly altered in the early development stages in HP. CONCLUSION: The high sugar accumulation in HP fruit was associated with up-regulation of SUS, SPS, TST, STP and ERD6L genes. The PEPCK, PEPC, NAD-MDH, NADP-IDH, GS and FBPase genes played important roles in acid synthesis and degradation in HP kumquat. These findings provide further insight into understanding the mechanisms underlying metabolism of sugars and organic acids in citrus. © 2021 Society of Chemical Industry.

(+/-)-Dievodialetins A-G: Seven pairs of enantiomeric coumarin dimers with anti-acetylcholinesterase activity from the roots of Evodia lepta Merr.

Seven pairs of undescribed enantiomeric bis-coumarins, (±)-dievodialetins A-G, were separated from the roots of Evodia lepta Merr. Two coumarin nuclei were linked via a 1,4-dimethyl4-vinylcyclohexene moiety in (±)-dievodialetins C-G. The structures of the undescribed compounds, including their absolute configurations were elucidated by spectroscopic analyses, X-ray diffraction, and computational calculations. In the biosynthetic pathways, these bis-coumarins were presumably derived from the precursors demethylsuberosin and 3-(3-methylbut-2-enyl)umbelliferone via a [4 + 2] Diels-Alder reaction. Besides, all compounds exhibited neuroprotective effects by inhibiting acetylcholinesterase (AChE) activity with IC50 values ranging from 7.3 to 12.1 nM and they also suppressed oxidative stress (MDA and SOD) and neuroinflammation (IL-1ß and IL-6).

7-Isopentenyloxycoumarin: What Is New across the Last Decade.

7-Isopentenyloxycoumarin is among the most widespread naturally occurring prenyloxy umbelliferone derivatives. This secondary metabolite of mixed biosynthetic origin has been typically isolated from plants belonging to several genera of the Rutaceae and Apiaceae families, comprising widely used medicinal plants and in general plants with beneficial effects on human welfare, as well as edible fruits and vegetables. Although known for quite a long time (more than 50 years), only in the last two decades has this natural compound been revealed to exert powerful and promising pharmacological properties, such as active cancer chemopreventive, antibacterial, antiprotozoal, antifungal, anti-inflammatory, neuroprotective, and antioxidant properties, among the activities best outlined in the recent literature. The aim of this comprehensive miniature review article is to detail the novel natural sources and the effects described during the last decade for 7-isopentenyloxycoumarin and what has been reported on the mechanisms of action underlying the observed biological activities of this oxyprenylated secondary metabolite. In view of the herein described data, suggestions on how to address future research on the abovementioned natural product and structurally related derivatives in the best ways according to the authors will be also provided.

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides.

Cyclic peptides are reported to have antibacterial, antifungal, and other bioactivities. Orbitides are a class of cyclic peptides that are small, head-to-tail cyclized, composed of proteinogenic amino acids and lack disulfide bonds; they are also known in several genera of the plant family Rutaceae. Melicope xanthoxyloides is the Australian rain forest tree of the Rutaceae family in which evolidine, the first plant cyclic peptide, was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, so to redress this we used tandem MS and de novo transcriptomics to rediscover evolidine and decipher its biosynthetic origin from a short precursor just 48 residues in length. We also identified another six M. xanthoxyloides orbitides using the same techniques. These peptides have atypically diverse C termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides, suggesting new cyclizing enzymes await discovery. We examined the structure of two of the novel orbitides by NMR, finding one had a definable structure, whereas the other did not. Mining RNA-seq and whole genome sequencing data from other species of the Rutaceae family revealed that a large and diverse family of peptides is encoded by similar sequences across the family and demonstrates how powerful de novo transcriptomics can be at accelerating the discovery of new peptide families.

Metabolite Profiling by Hyphenated Liquid Chromatographic Mass Spectrometric Technique (HPLC-DAD-ESI-Q-TOF-MS/MS) and Neurobiological Potential of Haplophyllum sahinii and H. vulcanicum Extracts.

In the current study, the ethanol extracts of flower, stem, and root parts of two endemic Turkish species, e. g., Haplophyllum sahinii O. Tugay & D. Ulukus and H. vulcanicum Boiss. & Heldr., were screened against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) associated with Alzheimer's disease as well as tyrosinase (TYR) linked to Parkinson's disease using ELISA microplate assay at 200 µg/mL. Among the extracts, the highest inhibition was caused by the stem extract of H. sahinii against BChE (IC50 =64.93±1.38 µg/mL). Consistently, all of the extracts were found to exert a selective inhibition towards BChE to some extent. It was only the root extract of H. vulcanicum that could inhibit AChE at low level (IC50 =203.18±5.33 µg/mL). None of the extracts displayed an inhibition over 50 % against TYR. Metabolite profiling of the extracts was achieved by a highly hyphenated liquid chromatographic mass spectrometric technique (HPLC-DAD-ESI-Q-TOF-MS/MS), which revealed the presence of furoquinoline (ß-fagarine, γ-fagarine) and amide (tubasenicine, tubacetine) alkaloids; furano- (rutamarin), pyrano- (xanthyletine), and geranyloxy coumarins; phenylpropanoid (secoisolariciresinol), arylnaphthalene (mono-O-acetyldiphyllin apioside), and dibenzylbutyrolactone (kusunokinin, haplomyrfolin) lignans. Several important differences were observed between the extracts analyzed. ß-Fagarine was the major alkaloid in H. vulcanicum, whereas γ-fagarine was present only in the roots of both Haplophyllum species; moreover, secoisolariciresinol and secoisolariciresinol dimethyl ether were the main lignans in the stems and flowers. This is the first study identifying ChE and TYR inhibitory effect and metabolic profiles of H. vulcanicum and H. sahinii.

Effects of leaf colorness, pigment contents and allelochemicals on the orientation of the Asian citrus psyllid among four Rutaceae host plants.

BACKGROUND: Asian citrus psyllid (ACP) is the primary vector responsible for the transmission of the phloem-limited bacteria Candidatus Liberibacter spp., associated with huanglongbing (HLB), which causes great loss to the citrus industry. Although the roles of leaf color and volatile compounds in the orientation of ACP have been proven, the quantification of color and allelochemicals in the host plant are kept unclear, especially in wild citrus germplasms. RESULTS: Chongyi wild mandarin significantly attracted more ACP than wild Hong Kong kumquat, 'Gannan zao' navel orange and orange jasmine did in the four-choice and olfactometer assays. The color parameters of the tender leaves from Chongyi wild mandarin and 'Gannan zao' were similar. The yellow color in both of them was less saturated than that of the other two plants species, but Chongyi wild mandarin had significant lower carotenoid content (P < 0.05). Notably metabolic profiling differences were observed among the healthy tender shoots from the four tested plants via UPLC-QQQ-MS and GC-MS analyses. Comparing with the other three plant species, 66 and 50 metabolites with significantly different contents in Chongyi wild mandarin were selected as UPLC-identified and GC-identified metabolites of interest (P < 0.05), respectively. Flavonoids accounted for a large group of secondary metabolites of interest, which may function as stimulants or repellents of ACP. Higher content of salicylic acid o-hexoside and lower content of (+)-jasmonic acid in Chongyi wild mandarin may lead to higher amount of methyl salicylate (an ACP attractant) and lower amount of trans-ocimene (an attractant to herbivores' natural enemies) as well as the suppression of JA-mediated wounding response. This kind of synergistic or antagonistic effect among the metabolites differentially accumulated in Chongyi wild mandarin made it a more attractive host plant to ACP. CONCLUSIONS: Less saturated yellow color, high amount of attractants, low amount of repellents and insensitivity of JA-mediated wounding response are the four possible reasons why Chongyi wild mandarin attracted more ACP. This work may shed light on the olfactory and visual response of ACP to wild citrus germplasm hosts, and suggest the feasibility of developing ACP attractants or repellents patterned on potential metabolites.

Evolution of phytochemical diversity in Pilocarpus (Rutaceae).

The evolution of phytochemical diversity and biosynthetic pathways in plants can be evaluated from a phylogenetic and environmental perspective. Pilocarpus Vahl (Rutaceae), an economically important medicinal plant in the family Rutaceae, has a great diversity of imidazole alkaloids and coumarins. In this study, we used phylogenetic comparative methods to determine whether there is a phylogenetic signal for chemical traits across the genus Pilocarpus; this included ancestral reconstructions of continuous and discrete chemical traits. Bioclimatic variables found to be associated with the distribution of this genus were used to perform OLS regressions between chemical traits and bioclimatic variables. Next, these regression models were evaluated to test whether bioclimatic traits could significantly predict compound concentrations. Our study found that in terms of compound concentration, variation is most significantly associated with adaptive environmental convergence rather than phylogenetic relationships. The best predictive model of chemical traits was the OLS regression that modeled the relationship between coumarin and precipitation in the coldest quarter. However, we also found one chemical trait was dependent on phylogenetic history and bioclimatic factors. These findings emphasize that consideration of both environmental and phylogenetic factors is essential to tease out the intricate processes in the evolution of chemical diversity in plants. These methods can benefit fields such as conservation management, ecology, and evolutionary biology.

Biogenic synthesis of iron oxide nanoparticles via Skimmia laureola and their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum.

Plant diseases are threat to global food security. The excessive use of agrochemicals is the leading cause of pesticides resistance and toxicity to beneficial life forms. The quest for innocuous and alternate antimicrobial agent is crucial in order to overcome the pathogen resistance and the birth of nanotech offers pledge to combat pathogenic organisms. In this study, a facile benign biogenic approach was adopted for the synthesis of biocompatible iron oxide nanoparticles (Fe2O3-NPs) via Skimmia laureola leaf extract and the synthesized nanoparticles were evaluated for their antibacterial efficacy against bacterial wilt pathogen Ralstonia solanacearum in vitro and in planta. Physico-chemical characterization of the synthesized nanoparticles was performed through UV-Visible spectroscopy, Fourier Transform Infrared Spectroscopy, X-Ray Diffraction, Energy Dispersive X-ray Spectroscopy and Scanning Electron Microscopy. The results revealed polydisperse nanoparticles in the size range of 56 nm to 350 nm. The culture media containing 6 mg/mL of Fe2O3-NPs dramatically inhibited the bacterial growth in vitro. Scanning electron microscopy revealed degenerative characteristics including degraded, shriveled and concentrated cell walls. Diseases severity was effectively reduced with 6% w/v of Fe2O3-NPs treated root zone in planta. Plant shoots, root length and fresh biomass were enhanced with Fe2O3-NPs treatments. The results indicated that the biosynthesized Fe2O3-NPs have the potential to control agriculturally important phytopathogen Ralstonia solanacearum in vitro and in planta.

Transcriptome analysis of the curry tree (Bergera koenigii L., Rutaceae) during leaf development.

The curry tree (Bergera koenigii L.) is a widely cultivated plant used in South Asian cooking. Next-generation sequencing was used to generate the transcriptome of the curry leaf to detect changes in gene expression during leaf development, such as those genes involved in the production of oils which lend the leaf its characteristic taste, aroma, and medicinal properties. Using abundance estimation (RSEM) and differential expression analysis, genes that were significantly differentially expressed were identified. The transcriptome was annotated with BLASTx using the non-redundant (nr) protein database, and Gene Ontology (GO) terms were assigned based on the top BLAST hit using Blast2GO. Lastly, functional enrichment of the assigned GO terms was analyzed for genes that were significantly differentially expressed. Of the most enriched GO categories, pathways involved in cell wall, membrane, and lignin synthesis were found to be most upregulated in immature leaf tissue, possibly due to the growth and expansion of the leaf tissue. Terpene synthases, which synthesize monoterpenes and sesquiterpenes, which comprise much of the curry essential oil, were found to be significantly upregulated in mature leaf tissue, suggesting that oil production increases later in leaf development. Enzymes involved in pigment production were also significantly upregulated in mature leaves. The findings were based on computational estimates of gene expression from RNA-seq data, and further study is warranted to validate these results using targeted techniques, such as quantitative PCR.

Immunostimulatory effect of kumquat (Fortunella crassifolia) and its constituents, ß-cryptoxanthin and R-limonene.

Natural killer (NK) cells play an important role in the innate immune system by eliminating cancer cells and virally infected cells. Aging and stress attenuate the activity of NK cells, thereby increasing the risk of various diseases. In this study, we demonstrated that the consumption of a small number of kumquats in an in vivo model could suppress elevated plasma corticosterone levels and reverse the decline in splenocyte cytotoxicity caused by restraint stress. Our results identified ß-cryptoxanthin (BCX) as an active kumquat component with a NK cell-activating effect, and R-limonene as an active component that mediates not only the anti-stress effect but also NK cell activation by oral administration. In addition, BCX, R-limonene, and R-limonene metabolites were found to enhance IFN-γ production in KHYG-1 cells, a human NK cell line. Collectively, our findings suggest that the ingestion of a few kumquats on a daily basis can help to combat stress and enhance NK cell activity.

The transcription factor FcWRKY40 of Fortunella crassifolia functions positively in salt tolerance through modulation of ion homeostasis and proline biosynthesis by directly regulating SOS2 and P5CS1 homologs.

Although some WRKYs have been characterized, regulatory roles of most WRKYs remain poorly understood. Herein, we elucidated function of FcWRKY40 from Fortunella crassifolia in salt tolerance via overexpression and virus-induced gene silencing (VIGS) and unraveled its target genes. Overexpression of FcWRKY40 enhanced salt tolerance in transgenic tobacco and lemon, while silencing of FcWRKY40 increased salt susceptibility. Homolog genes of Salt Overly Sensitive 2 (SOS2) and Δ-1-pyrroline-5-carboxylate synthetase 1 (P5CS1) were dramatically up-regulated in transgenic lemon but down-regulated in VIGS line. Consistently, transgenic lemon displayed lower Na+ and higher proline concentrations, whereas the silenced line accumulated more Na+ but less proline. Treatment of transgenic lemon with 24-epi-brassinolide compromised salt tolerance, while supply of exogenous proline partially restored salt tolerance of the VIGS line. FcWRKY40 specifically binds to and activates promoters of FcSOS2 and FcP5CS1. FcWRKY40 was up-regulated by ABA and salt, and confirmed as a target of ABA-responsive element binding factor 2 (FcABF2). Moreover, salt treatment up-regulated FcABF2 and FcP5CS1, and elevated proline concentrations. Taken together, our findings demonstrate that FcWRKY40 participates in the ABA signaling pathway and as a positive regulator functions in salt tolerance by regulating genes involved in ion homeostasis and proline biosynthesis.

A review on bio-synthesized zinc oxide nanoparticles using plant extracts as reductants and stabilizing agents.

In the age of technology, nanoparticles have proven to be one of the essential needs for development. These nanoparticles have the potential to be used for a wide variety of applications, thereby, development in improving the quality of nanoparticles, to make them more application specific, is still under research. In this regard, an important point to note is that the procedures employed in synthesizing nanoparticles require to be cost-effective and less-steps involved and have an additional advantage, i.e. they should be eco-friendly. This means that the synthesis procedure needs avoiding the use of harmful chemicals, and negligible generation of any noxious by-products. The green synthesis (biosynthesis) method employs simple procedures, easily available raw materials and ambiance for the synthesis process, where the precursors used are safe, with minute possibility for the production of harmful by-products. Considering these advantages, the current review includes a brief description on the various chemical and physical synthesis method of zinc oxide (ZnO) nanoparticles with emphasis on the biosynthesis of ZnO nanoparticles using plant extracts (and briefly microbes), the phytochemicals present in the plant extracts, the plausible mechanisms involved in the formation of ZnO nanoparticles and applications of the as-synthesized ZnO nanoparticles as photocatalysts and microbial inhibitors.

Alkaloid diversity in the leaves of Australian Flindersia (Rutaceae) species driven by adaptation to aridity.

The genus Flindersia (Rutaceae) comprises 17 species of mostly Australian endemic trees. Although most species are restricted to rainforests, four have evolved to grow in semi-arid and arid environments. In this study, the leaf alkaloid diversity of rainforest and semi-arid/arid zone adapted Australian Flindersia were compared by LC/MS-MS and NMR spectroscopy. Contrary to expectations, Flindersia alkaloid diversity was strongly correlated with environmental aridity, where species predominating in drier regions produced more alkaloids than their wet rainforest congenerics. Rainforest species were also more chemically similar to each other than were the four semi-arid/arid zone species. There was a significant relationship between the presence of alkaloid structural classes and phylogenetic distance, suggesting that alkaloid profiles are influenced by both genetic and environmental factors. The results suggest that the radiation of Flindersia species out of the rainforest and into drier environments has promoted the evolution of unique alkaloid diversity. Plants growing in arid and semi-arid regions of Australia may represent an untapped source of undescribed specialised metabolites.

Biosynthesis, characterization and antimicrobial activities of zinc oxide nanoparticles from leaf extract of Glycosmis pentaphylla (Retz.) DC.

Biosynthesized nanoparticles have an incredible application in biomedicine owing to its simplicity, eco-friendly properties and low cost. The present study aims to determine the green synthesized zinc oxide nanoparticles from methanolic leaf extract of Glycosmis pentaphylla. The synthesized nanoparticles were characterized using UV-VIS Spectroscopy, Fluorescence spectrometer, FT-IR, XRD, SEM with EDAX and TEM. The confirmations of synthesized nanoparticles were characterized by peak at 351 and 410 nm in the UV-VIS spectrum and photoluminescence spectrum respectively. FT-IR studies revealed the functional group of the nanoparticles. The XRD data showed the crystalline nature of the nanoparticles and EDAX measurements indicated the 20.70% of highly pure zinc oxide metal. The morphological characterization of synthesized zinc oxide nanoparticles was analyzed by SEM and TEM and size of the particles were ranging from 32 to 36 nm. The synthesized zinc oxide nanoparticles exhibited interesting antimicrobial activity against pathogenic organisms. In addition, this is the first report on leaf mediated synthesis of zinc oxide (ZnO) nanoparticles from Glycosmis pentaphylla.