ApCPS2 contributes to medicinal diterpenoid biosynthesis and defense against insect herbivore in Andrographis paniculata.

Kalmegh (Andrographis paniculata) spatiotemporally produces medicinally-important ent-labdane-related diterpenoids (ent-LRDs); andrographolide (AD), 14-deoxy-11,12-didehydroandrographolide (DDAD), neoandrographolide (NAD). ApCPS1 and ApCPS2, the ent-copalyl pyrophosphate (ent-CPP)-producing class II diterpene synthases (diTPSs) were identified, but their contributions to ent-CPP precursor supply for ent-LRD biosynthesis were not well understood. Here, we characterized ApCPS4, an additional ent-CPP-forming diTPS. Further, we elucidated in planta function of the ent-CPP-producing diTPSs (ApCPS1,2,4) by integrating transcript-metabolite co-profiles, biochemical analysis and gene functional characterization. ApCPS1,2,4 localized to the plastids, where diterpenoid biosynthesis occurs in plants, but ApCPS1,2,4 transcript expression patterns and ent-LRD contents revealed a strong correlation of ApCPS2 expression and ent-LRD accumulation in kalmegh. ApCPS1,2,4 upstream sequences differentially activated ß-glucuronidase (GUS) in Arabidopsis and transiently-transformed kalmegh. Similar to higher expression of ApCPS1 in kalmegh stem, ApCPS1 upstream sequence activated GUS in stem/hypocotyl of Arabidopsis and kalmegh. However, ApCPS2,4 upstream sequences weakly activated GUS expression in Arabidopsis, which was not well correlated with ApCPS2,4 transcript expression in kalmegh tissues. Whereas, ApCPS2,4 upstream sequences could activate GUS expression at a considerable level in kalmegh leaf and roots/calyx, respectively, suggesting the involvement of transcriptional regulator(s) of ApCPS2,4 that might participate in kalmegh-specific diterpenoid pathway. Interestingly, ApCPS2-silenced kalmegh showed a drastic reduction in AD, DDAD and NAD contents and compromised defense against insect herbivore Spodoptera litura. However, ent-LRD contents and herbivore defense in ApCPS1 or ApCPS4-silenced plants remained largely unaltered. Overall, these results suggested an important role of ApCPS2 in producing ent-CPP for medicinal ent-LRD biosynthesis and defense against insect herbivore.

Failure of methanol detoxification in pests confers broad spectrum insect resistance in PME overexpressing transgenic cotton.

Methanol is noxious to insect pests, but most plants do not make enough of it to shield themselves from encroaching insects. Methanol emission is known to increase in the instance of herbivory. In the current study, we showed that Aspergillus niger pectin methylesterase over-expression increases methanol emission and confers resistance to polyphagous insect pests on transgenic cotton plants by impeding the possible methanol detoxification pathways. Transgenic plants emitted ∼11 fold higher methanol displaying insect mortality of 96% and 93% in Helicoverpa armigera and Spodoptera litura, respectively. The larvae were unable to survive and finish their life cycle and the surviving larvae exhibited severe growth retardation. Insects try to detoxify methanol via catalase, carboxylesterase and cytochrome P450 monooxygenase enzymes, amongst which cytochrome P450 plays a major role in oxidizing methanol to formaldehyde and formaldehyde to formic acid, which is broken down into carbon dioxide and water. In our study, catalase and esterase enzymes were found to be upregulated, but cytochrome P450 monooxygenase levels were not much affected. Leaf disc assays and In-planta bioassays also showed 50-60% population reduction in the sap sucking pests, such as Bemisia tabaci and Phenacoccus solenopsis. These findings imply that elevated methanol emissions confer resistance in plants against chewing and sap-sucking pests by tampering the methanol detoxification pathways. Such mechanism will be useful in imparting expansive resistance against pests in plants.

Giant Pleomorphic Adenomas of Parotid Gland: a Case Series.

The parotid gland is the largest salivary gland in the body. Pleomorphic adenomas (PA) are most common benign tumors of parotid gland. If left untreated, they can gradually attain the size which can weigh several kilograms. This paper highlights a case series of 15 cases diagnosed as giant pleomorphic adenoma arising in the parotid gland along with their management. A hospital based, observational study of 15 patients of PA of the parotid gland, > 10 cm in its widest dimension considered as giant, done at tertiary care ENT hospital over period of 9 years. The age of the patients ranged from 30 to 81 years with mean age 50.33 ± 15.30 years. There were 5(33.33%) males and 10(66.67%) females. The time duration of having tumor ranged from 5 to 20 years with mean duration of 10.4 ± 4.17 years and the largest tumor was 25 cm in its largest diameter. Mean weight of tumors was 2.72 ± 0.52 kg. One out of fifteen cases was detected as malignant in nature. All cases were treated by surgical excision without any complications. Pleomorphic adenoma of parotid gland can assume a giant proportion when timely surgical intervention is not done. We can build up confidence and hope for life in patients after complete surgical excision of giant PA.

A prospective longitudinal study of clinical outcome and quality of life assessment in patients with chronic rhinosinusitis after functional endoscopic sinus surgery using sino nasal outcome test-22.

Chronic rhinosinusitis (CRS) significantly affect the quality of life (QoL) of patients. The study was conducted in CRS patients who were treated with functional endoscopic sinus surgery (FESS) after failure of medical treatment to analyze clinical outcome using prospectively collected data through a symptom-based rhinosinusitis outcome measure, the Sino-nasal Outcome Test-22 (SNOT-22). The aim of the study was to evaluate and compare the QoL in patients of chronic rhinosinusitis pre-operative and after FESS by SNOT-22. The prospective study was conducted on 40 patients of chronic rhinosinusitis with or without nasal polyposis. Demographic, clinical, diagnostic nasal endoscopy and radiological findings were recorded. Visual analogue scoring and SNOT-22 questionnaire scoring were done preoperatively and at 3rd and 6th months post-operatively. These scores were compared and a value of p < 0.01 was considered statistical significant. Nasal obstruction (80%) was the most commonly reported disabling condition followed by rhinorrhea (75%), facial pain-pressure (72.5%), headache and sneezing. The mean preoperative nasal endoscopy score was 8.08 ± 3.65. The mean preoperative Lund Mackay CT scan score was 11.725 ± 3.64. The mean preoperative SNOT-22 score was 46.25 ± 20.44. After FESS, nasal discharge was improved in 86% patients. Average VAS scores showed significant postoperative improvement at 6 months (p < 0.01). The mean postoperative diagnostic nasal endoscopy score improved to 2.80 ± 1.64 at 6 months (p < 0.01). The mean postoperative SNOT-22 scores decreased at postoperative follow up visits at 3 and 6 months to 14.58 ± 4.90 at 3 months and 22.38 ±7.93 at 6 months (p < 0.01). CRS patient refractory to medical treatment showed statistical significant improvement after FESS. The SNOT-22 scoring was easy to use scoring used for QoL assessment showed significant improvement after FESS.

A Prospective Longitudinal Study to Evaluate the Patency of the Aditus ad Antrum in Chronic Otitis Media Mucosal Type.

Chronic otitis media is defined as the chronic inflammation of the mucoperiosteal lining lining of the middle ear cleft which presents with recurrent ear discharge through tympanic membrane perforation. The study was planned to evaluate the patency of the aditus ad antrum in cases of chronic otitis media mucosal type, and its correlation with various direct and indirect indicators of blocked aditus. The prospective longitudinal study was conducted on 100 patients with chronic otitis media mucosal type. The commonest complaint of patients was ear discharge (100%) followed by hearing impairment in 72% patients.Tympanoplasty with antrotomy was performed. The patency and dimensions of the aditus ad antrum were assessed by saline water test. In 41% cases saline test showed blockade, on further drilling patency achieved in 27 cases with minimal drilling and dissection of mucosa with or without fibrotic mucosal bands. In rest 14 cases widening of aditus and atticotomy was required to achieve patency. Out of these nine were having associated tympanosclerosis and five were having edematous mucosa. Ossicular necrosis was seen on 18 cases. Presence of myringosclerosis and polypoidal edematous mucosa increases the probability of an obstructed aditus ad antrum. Mastoid antrostomy and water test for patency can be performed without additional cost and risk to the patient in minimal time and can be considered as the surest indicator of patency of aditus ad antrum.

A Prospective Randomized Study to Compare Surgical Outcome Using Wet and Dry Temporalis Fascia Graft in Type I Tympanoplasty.

Temporalis fascia is being used as graft material for tympanic membrane repair for over six decades. Though cartilage with or without perichondrium is also being used for different situations, still temporalis fascia graft is being considered as an ideal graft for tympanoplasty. Earlier the dried temporalis fascia was used as a graft but now freshly taken wet temporalis fascia is increasingly being used as a more 'physiological/ live' graft. The present study was planned to compare the results of use of wet and dry temporalis fascia in chronic otitis media mucosal disease. The prospective study was conducted on 100 patients of chronic suppurative otitis media mucosal disease. Chronic ear survey questionnaire were filled to note the impact on quality of life. Otoscopic, oto-endoscopic and examination under microscope performed. Patients were allocated into two groups alternately. Freshly taken wet temporalis graft was used in group I whereas dry temporalis fascia was used in group II. Hearing evaluation by pure tone audiogram was done preoperatively and at 6th months post-operatively. Pre-operative and post-operative hearings in both the groups were compared. Air bone gap closures after surgery in between both the groups were compared to see for any statistical difference. We found similar graft uptake in both the groups after 6 months of surgery. The air bone gap closure in group I was 10.80 ± 5.74 and group II was 9.28 ± 4.81, there was no significant difference. However senior authors noted that in some of the cases in group I with fresh wet graft placement neo-tympanum formation was noticed early showing good circumferential leash of blood vessels all around signifying early vascularisation.

Molecular Characterization, Evolutionary Analysis, and Expression Profiling of BOR Genes in Important Cereals.

Boron (B) is an essential micronutrient of plants. Plants grapple with a narrow range of B between its toxicity and deficiency. B homeostasis mechanism is required to rescue plants from such a quagmire. B transporters are specialized proteins involved in the homeostasis of B. In the present study, a total of 29 BOR genes were identified in five major cereals, including three BORs in each Brachypodium distachyon and Sorghum bicolor, four in Oryza sativa, six in Zea mays, and 13 in Triticum aestivum. Multiple sequence alignments, domain structure analyses, and phylogenetic analysis indicated the conserved nature of the BOR protein family. Duplication events and Ka/Ks analysis of TaBORs showed the role of segmental duplication events and purifying selection in the expansion of the BOR family in T. aestivum. Furthermore, in silico expression and co-expression analyses under biotic and abiotic stress conditions depicted their involvement in combating such conditions. Moreover, qRT-PCR of TaBORs in B treatment suggested the roles of BOR genes in B stress management. The present study hints at the conserved nature of BOR proteins and their different aspects. The study will lay down a way for several crop improvement programs.

Identification and validation of the wound and insect bite early inducible promoter from Arabidopsis thaliana.

A wound-inducible promoter facilitates the regulated gene expression at the targeted site during the time of mechanical stress or infestation by the pathogen. The present work has aimed to identify a wound-inducible promoter that expresses at early time points preceding wound-stress treatment in Arabidopsis thaliana. The computational analysis of microarray data (GSE5627) resulted in the identification of five early inducible genes, viz., AT1G17380, AT1G80440, AT2G43530, AT3G48360, and AT5G13220. The RT-PCR analysis showed AT5G13220 (JASMONATE-ASSOCIATED 1) gene induced at a significantly higher level post 30 min of wounding. Thus, the promoter of the highly induced and early expressed wound-inducible gene, AT5G13220 (named PW220), was characterized by fusing with ß-glucuronidase (gusA) reporter or Cry1EC genes. The fluorometric analysis and histochemical staining of the gusA gene and quantitative estimation of Cry1EC protein in Nicotiana tabacum transgenic lines confirmed wound-induced expression characteristic of the selected promoter. Insect bioassay suggested that wound-inducible and constitutive expression of Cry1EC protein in transgenic lines showed a similar level of protection against different instar Spodoptera litura larvae. Furthermore, we identified that abscisic acid influenced the wound-specific expression of the selected PW220 promoter in the transgenic lines, which correlates with the presence of conserved cis-regulatory elements associated with dehydration and abscisic acid responses. Altogether, our results suggested that the wound-inducible promoter PW220 provides an excellent alternative for developing insect-tolerant transgenic crops in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03143-0.

Unravelling Cotton Nonexpressor of Pathogenesis-Related 1(NPR1)-Like Genes Family: Evolutionary Analysis and Putative Role in Fiber Development and Defense Pathway.

The nonexpressor of pathogenesis-related 1 (NPR1) family plays diverse roles in gene regulation in the defense and development signaling pathways in plants. Less evidence is available regarding the significance of the NPR1-like gene family in cotton (Gossypium species). Therefore, to address the importance of the cotton NPR1-like gene family in the defense pathway, four Gossypium species were studied: two tetraploid species, G.hirsutum and G. barbadense, and their two potential ancestral diploids, G. raimondii and G. arboreum. In this study, 12 NPR1-like family genes in G. hirsutum were recognized, including six genes in the A-subgenome and six genes in the D-subgenome. Based on the phylogenetic analysis, gene and protein structural features, cotton NPR-like proteins were grouped into three different clades. Our analysis suggests the significance of cis-regulatory elements in the upstream region of cotton NPR1-like genes in hormonal signaling, biotic stress conditions, and developmental processes. The quantitative expression analysis for different developmental tissues and fiber stages (0 to 25 days post-anthesis), as well as salicylic acid induction, confirmed the distinct function of different cotton NPR genes in defense and fiber development. Altogether, this study presents specifications of conservation in the cotton NPR1-like gene family and their functional divergence for development of fiber and defense properties.

Molecular characterization of ascorbate peroxidase (APX) and APX-related (APX-R) genes in Triticum aestivum L.

Ascorbate peroxidases (APXs) are heme-dependent H2O2 scavenging enzymes involved in myriad biological processes. Herein, a total of 21 TaAPX and six TaAPX-R genes were identified from the A, B and D sub-genomes of Triticum aestivum L. The occurrence of three paralogous gene pairs with unequal evolutionary rate suggested functional divergence. The phylogenetic analysis formed four distinct clades having conserved gene and protein architecture, and sub-cellular localization. The tertiary structure analysis revealed the presence of helices and coils and residues involved in ligand binding. Transcriptional profiling of each TaAPX and TaAPX-R gene suggested their specific role during development and stress response. Modulated transcript expression and APX enzyme activity during various stress conditions indicated their role in stress response. Interaction analyses suggested their association with other genes, miRNAs and various legends. The present study reported numerous features of these genes, and may provide a platform for their detailed functional characterization in future studies.

Distinct defensive activity of phenolics and phenylpropanoid pathway genes in different cotton varieties toward chewing pests.

Identifying the maximum level of inherent defense against harmful insects in natural variation among wild lineages of crop plants may result in high yield tolerant varieties and reducing use of chemical insecticides. However, knowledge of natural cotton genotypes with high insect-resistance is still indistinguishable at the biochemical or molecular level. In the present study, different cultivated Gossypium hirsutum varieties were evaluated for their inherent insect-tolerance against two major cottons chewing pests. The insect bio-assay identified two tolerant and one susceptible cotton varieties. The study demonstrates difference in phenolic acids, proanthocyanidin and tannin accumulation in tolerant and susceptible varieties. The post-infestation of chewing pests increases transcript level of the phenylpropanoid pathway genes were detected in tolerant varieties as compared to the susceptible varieties. Altogether, chewing pest-tolerance level in cotton varieties is the cumulative effect of enhanced phenylpropanoid pathway genes expression and secondary metabolite leading to defense responses to conventional host plant.

Post Parotidectomy Quality of Life in Patients with Benign Parotid Neoplasm: A Prospective Study.

Parotid tumours are not uncommon. The management is surgical for benign and malignant parotid neoplasm. Due to the location of parotid gland and its intricate relationship with facial nerve, cosmetic and functional outcomes after parotid surgery are extremely important. Objectives of the study were to analyse facial nerve functions with emphasis on the quality of life of patients undergoing surgery for parotid neoplasm. A prospective study was conducted on patients presented with parotid neoplasm and undergone parotid surgery. Patient with malignant neoplasm were excluded. 30 patients with benign parotid neoplasm in final histopathology were included in the study. Post operative assessment of facial nerve was done using postparotidectomy facial nerve grading system. Symptom-specific QOL was assessed with the parotidectomy outcome inventory-8. Aesthetic outcome was evaluated with an ordinal scale. Posterior belly of digastric muscle and tragal pointer were the commonest landmark used for facial nerve identification. Temporary facial nerve dysfunction was present in six (20%) patients with marginal mandibular branch most commonly involved. 96% of the female patients and 91% of the male patients rated the cosmetic result as good or very good. A statistically significant difference is noted between superficial parotidectomy and total Parotidectomy for cosmetic outcome and sensory impairment. We noted that changed appearance due to resection of the parotid gland and scar and sensory impairment in the area affect the quality of life of patients and such affect are more after total conservative parotidectomy.

De novo characterization of Phenacoccus solenopsis transcriptome and analysis of gene expression profiling during development and hormone biosynthesis.

The cotton mealybug Phenacoccus solenopsis is a devastating pest of cotton causing tremendous loss in the yield of crops each year. Widespread physiological and biological studies on P. solenopsis have been carried out, but the lack of genetic information has constrained our understanding of the molecular mechanisms behind its growth and development. To understand and characterize the different developmental stages, RNA-Seq platform was used to execute de-novo transcriptome assembly and differential gene expression profiling for the eggs, first, second, third instar and adult female stages. About 182.67 million reads were assembled into 93,781 unigenes with an average length of 871.4 bp and an N50 length of 1899 bp. These unigenes sequences were annotated and classified by performing NCBI non-redundant (Nr) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Clusters of Orthologous Groups (COG), Gene ontology (GO), the Swiss-Prot protein database (Swiss-Prot), and nearest related organism Acyrthosiphon pisum (pea aphid) database. To get more information regarding the process of metamorphosis, we performed a pairwise comparison of four developmental stages and obtained 29,415 differentially expressed genes. Some of the differentially expressed genes were associated with functional protein synthesis, anti-microbial protection, development and hormone biosynthesis. Functional pathway enrichment analysis of differentially expressed genes showed the positive correlation with specific physiological activities of each stage, and these results were confirmed by qRT-PCR experiments. This study gives a valuable genomics resource of P. solenopsis covering all its developmental stages and will promote future studies on biological processes at the molecular level.

Reference genes validation in Phenacoccus solenopsis under various biotic and abiotic stress conditions.

Real-time PCR (RT-qPCR) expression analysis is a powerful analytical technique, but for normalization of data requires the use of stable reference genes. However, suitable reference genes are still not known in the case of Phenacoccus solenopsis under variable experimental treatments. The present study focused on the identification of stable housekeeping genes as a reference for analysis under different abiotic and biotic factors in P. solenopsis. We analyzed the relative expression of six commonly used candidate reference genes in different developmental stages, host-feeding assay, temperature treatments and field distribution conditions. Expression stabilities were analyzed by geNorm, NormFinder, and RefFinder. Under developmental and field distribution conditions, ß-Tubulin was found to be most stable reference genes followed by rpl32 and α-Tubulin. In the case host feeding treatment conditions, ß-Tubulin and α-tubulin identified to be the most stable reference genes, while in temperature stress, a combination of α-Tubulin and rpl32 found to be suitable for normalizing the RT-qPCR data. Further, the above-identified genes were validated using RT-qPCR based gene expression analysis of four objective genes namely, Myoinhibitory peptides (MIPs), Zinc_metalloprotease (Zn_Mp), fatty acid synthase (fas) and alpha-glucosidase. Identified reference genes will facilitate gene expression studies in future under different stress treatments in P. solenopsis.

A novel arsenic methyltransferase gene of Westerdykella aurantiaca isolated from arsenic contaminated soil: phylogenetic, physiological, and biochemical studies and its role in arsenic bioremediation.

Elevated arsenic concentration in the environment and agricultural soil is a serious concern to crop production and human health. Among different detoxification mechanisms, the methylation of arsenic is a widespread phenomenon in nature. A number of microorganisms are able to methylate arsenic, but less is known about the arsenic metabolism in fungi. We identified a novel arsenic methyltransferase (WaarsM) gene from a soil fungus, Westerdykella aurantiaca. WaarsM showed sequence homology with all known arsenic methyltransferases having three conserved SAM binding motifs. The expression of WaarsM enhanced arsenic resistance in E. coli (Δars) and S. cerevisiae (Δacr2) strains by biomethylation and required endogenous reductants, preferably GSH, for methyltransferase activity. The purified WaarsM catalyzes the production of methylated arsenicals from both AsIII and AsV, and also displays AsV reductase activity. It displayed higher methyltransferase activity and lower KM 0.1945 ± 0.021 mM and KM 0.4034 ± 0.078 mM for AsIII and AsV, respectively. S. cerevisiae (Δacr2) cells expressing WaarsM produced 2.2 ppm volatile arsenic and 0.64 ppm DMA(v) with 0.58 ppm volatile arsenicals when exposed to 20 ppm AsV and 2 ppm AsIII, respectively. Arsenic tolerance in rice after co-culture with genetically engineered yeast suggested its potential role in arsenic bioremediation. Thus, characterization of WaarsM provides a potential strategy to reduce arsenic concentration in soil with reduced arsenic accumulation in crops grown in arsenic contaminated areas, and thereby alleviating human health risks.

Transcriptome sequencing of a thalloid bryophyte; Dumortiera hirsuta (Sw) Nees: assembly, annotation, and marker discovery.

Bryophytes are the first land plants but are scarcely studied at the molecular level. Here, we report transcriptome sequencing and functional annotation of Dumortiera hirsuta, as a representative bryophyte. Approximately 0.5 million reads with ~195 Mb data were generated by sequencing of mRNA using 454 pyrosequencer. De novo assembly of reads yielded 85,240 unigenes (12,439 contigs and 72,801 singletons). BlastX search at NCBI-NR database showed similarity of 33,662 unigenes with 10-(10) e-value. A total of 23,685 unigenes were annotated at TAIR10 protein database. The annotated unigenes were further classified using the Gene Ontology. Analysis at Kyoto Encyclopedia of Genes and Genomes pathway database identified 95 pathways with significant scores, among which metabolic and biosynthesis of secondary metabolite were the major ones. Phenylpropanoid pathway was elucidated and selected genes were characterized by real time qPCR. We identified 447 transcription factors belonging to 41 families and 1594 eSSRs in 1479 unigenes. D. hirsuta unigenes showed homology across the taxa from algae to angiosperm indicating their role as the connecting link between aquatic and terrestrial plants. This could be a valuable genomic resource for molecular and evolutionary studies. Further, it sheds light for the isolation and characterization of new genes with unique functions.

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1-8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.

Evaluation of total phenolic compounds and insecticidal and antioxidant activities of tomato hairy root extract.

Tomatoes are one of the most consumed crops in the whole world because of their versatile importance in dietary food as well as many industrial applications. They are also a rich source of secondary metabolites, such as phenolics and flavonoids. In the present study, we described a method to produce these compounds from hairy roots of tomato (THRs). Agrobacterium rhizogenes strain A4 was used to induce hairy roots in the tomato explants. The Ri T-DNA was confirmed by polymerase chain reaction amplification of the rolC gene. Biomass accumulation of hairy root lines was 1.7-3.7-fold higher compared to in vitro grown roots. Moreover, THRs efficiently produced several phenolic compounds, such as rutin, quercetin, kaempferol, gallic acid, protocatechuic acid, ferulic acid, colorogenic acid, and caffeic acid. Gallic acid [34.02 µg/g of dry weight (DW)] and rutin (20.26 µg/g of DW) were the major phenolic acid and flavonoid produced by THRs, respectively. The activities of reactive oxygen species enzymes (catalase, ascorbate peroxidase, and superoxide dismutase) were quantified. The activity of catalase in THRs was 0.97 ± 0.03 mM H2O2 min(-1) g(-1), which was 1.22-fold (0.79 ± 0.09 mM H2O2 min(-1) g(-1)) and 1.59-fold (0.61 ± 0.06 mM H2O2 min(-1) g(-1)) higher than field grown and in vitro grown roots, respectively. At 100 µL/g concentration, the phenolic compound extract caused 53.34 and 40.00% mortality against Helicoverpa armigera and Spodoptera litura, respectively, after 6 days. Surviving larvae of H. armigera and S. litura on the phenolic compound extract after 6 days showed 85.43 and 86.90% growth retardation, respectively.

Enhanced methanol production in plants provides broad spectrum insect resistance.

Plants naturally emit methanol as volatile organic compound. Methanol is toxic to insect pests; but the quantity produced by most of the plants is not enough to protect them against invading insect pests. In the present study, we demonstrated that the over-expression of pectin methylesterase, derived from Arabidopsis thaliana and Aspergillus niger, in transgenic tobacco plants enhances methanol production and resistance to polyphagous insect pests. Methanol content in the leaves of transgenic plants was measured using proton nuclear spectroscopy (1H NMR) and spectra showed up to 16 fold higher methanol as compared to control wild type (WT) plants. A maximum of 100 and 85% mortality in chewing insects Helicoverpa armigera and Spodoptera litura larvae was observed, respectively when fed on transgenic plants leaves. The surviving larvae showed less feeding, severe growth retardation and could not develop into pupae. In-planta bioassay on transgenic lines showed up to 99 and 75% reduction in the population multiplication of plant sap sucking pests Myzus persicae (aphid) and Bemisia tabaci (whitefly), respectively. Most of the phenotypic characters of transgenic plants were similar to WT plants. Confocal microscopy showed no deformities in cellular integrity, structure and density of stomata and trichomes of transgenic plants compared to WT. Pollen germination and tube formation was also not affected in transgenic plants. Cell wall enzyme transcript levels were comparable with WT. This study demonstrated for the first time that methanol emission can be utilized for imparting broad range insect resistance in plants.

Pectin methylesterase of Datura species, purification, and characterization from Datura stramonium and its application.

Pectin methylesterases (PME; EC 3.1.1.11) involved in de-esterification of pectin and have applicability in food, textiles, wines, pulp, and paper industries. In the present study, we compared PME activity of different parts of 3 Datura species and found that fruit coat showed maximum PME activity followed by leaf and seed. PME from leaves of D. stramonium (DsPME) was purified and characterized. DsPME showed optimum activity at 60 °C and pH 9 in the presence of 0.3 M NaCl. DsPME was stable at 70 °C and retained more than 40% activity after 60 min of incubation. However, enzyme activity completely abolished at 80 after 5 min of incubation. It follows Michaelis-Menten enzyme kinetics. Km and Vmax with citrus pectin were 0.008 mg/ml and 16.96 µmol/min, respectively. DsPME in combination with polygalactourenase (PGA) increased the clarity of orange, apple, pomegranate and pineapple juices by 2.9, 2.6, 2.3, and 3.6 fold, respectively in comparison to PGA alone. Due to very high de-esterification activity, easy denaturation and significant efficacy in incrementing clarification of fruit juice makes DsPME useful for industrial application.