Histochemical, metabolic and ultrastructural changes in leaf patelliform nectaries explain extrafloral nectar synthesis and secretion in Clerodendrum chinense.

BACKGROUND AND AIMS: Extrafloral nectaries are nectar-secreting structures present on vegetative parts of plants which provide indirect defences against herbivore attack. Extrafloral nectaries in Clerodendrum chinense are patelliform-shaped specialized trichomatous structures. However, a complete understanding of patelliform extrafloral nectaries in general, and of C. chinense in particular, has not yet been established to provide fundamental insight into the cellular physiological machinery involved in nectar biosynthesis and secretory processes. METHODS: We studied temporal changes in the morphological, anatomical and ultrastructural features in the architectures of extrafloral nectaries. We also compared metabolite profiles of extrafloral nectar, nectary tissue, non-nectary tissue and phloem sap. Further, both in situ histolocalization and normal in vitro activities of enzymes related to sugar metabolism were examined. KEY RESULTS: Four distinct tissue regions in the nectar gland were revealed from histochemical characterization, among which the middle nectariferous tissue was found to be the metabolically active region, while the intermediate layer was found to be lipid-rich. Ultrastructural study showed the presence of a large number of mitochondria along with starch-bearing chloroplasts in the nectariferous region. However, starch depletion was noted with progressive maturation of nectaries. Metabolite analysis revealed compositional differences among nectar, phloem sap, nectary and non-nectary tissue. Invertase activity was higher in secretory stages and localized in nectariferous tissue and adjacent region. CONCLUSIONS: Our study suggests extrafloral nectar secretion in C. chinense to be both eccrine and merocrine in nature. A distinct intermediate lipid-rich layer that separates the epidermis from nectary parenchyma was revealed, which possibly acts as a barrier to water flow in nectar. This study also revealed a distinction between nectar and phloem sap, and starch could act as a nectar precursor, as evidenced from enzymatic and ultrastructural studies. Thus, our findings on changing architecture of extrafloral nectaries with temporal secretion revealed a cell physiological process involved in nectar biosynthesis and secretion.

Light spectra manipulation stimulates growth, specialized metabolites and nutritional quality in Anethum graveolens.

Light-Emitting Diodes (LED) play a major role in manipulating light spectra that helps in regulating the growth and specialized metabolite synthesis relevant to the plant defence system. In this study, we assessed photosynthetic performance, phytonutrients, and anatomical variations of an aromatic herb Anethum graveolens (also known as dill), grown under various combinations of LED lights viz. red (100R:0B), red:blue (50R:50B); blue (0R:100B) and warm white (WW, served as control). Exposure to 0R:100B LED lights led to the tallest stem height, whereas, the number of leaves were highest under 50R:50B LED lights. The photosynthetic performance was observed to be highest under 50R:50B LED lights. HPLC analysis revealed chlorogenic acid and rosmarinic acid as the major phenolic compounds accumulated under different spectral irradiations. The highest chlorogenic acid content was observed in 50R:50B LED treated dill plants, while 100R:0B light showed the highest accumulation of rosmarinic acid. Dill plants grown under 50R:50B light displayed a relatively higher content of volatile compounds including, myristicin (phenylpropene), psi-limonene, and α-phellandrene (monoterpenoids). Expression analyses of candidate genes of phenylpropanoid and monoterpenoid biosynthetic pathways showed good correlations with the enhanced phenolic compounds and monoterpenes detected under appropriate light treatments. Further, the stem anatomy revealed higher vascularization under the influence of 0R:100B LED lights, whereas, intense histochemical localization of specialized metabolites could be correlated with enhanced accumulation of phenolic compounds and terpenoids observed in this study. Taken together, these studies suggest that proper combinations of blue and red spectra of light could play important role to augment the growth and phytochemical characteristics of dill, thus improving its value addition in the food industry.

Spectral Light Treatment Influenced Morpho-Physiological Properties and Carvacrol Accumulation in Indian Borage.

Light emitting diodes (LEDs) as an alternative light source for plants had shown to enhance the plant material quality. Indian borage or Plectranthus amboinicus (Lour.) Spreng, a medicinal herb produces carvacrol as the major volatile organic compound (VOC). Histolocalization of VOCs and expression pattern of the terpenoid biosynthesis genes after spectral light treatment is not yet reported in P. amboinicus. This work investigated the morpho-physiological, biochemical and transcriptional responses towards red, green, blue, warm white and red-blue (RB, 1:1) LEDs treatment at 40 ± 5 µmol m-2 s-1 light intensity after 40 days. Maximal growth index (GI), leaf fresh weight and dry weight were obtained in RB (1:1) treated plants. There was one-fold increase in phenolics content and 2.5-fold increase in antioxidant activity in comparison to warm white. High quantity of terpenes and phenolics deposition were observed in the glandular trichomes of RB (1:1). Maximum carvacrol accumulation (14.45 µmol g-1 FW) was also detected in RB (1:1). The transcript levels of early terpene biosynthesis genes PaDXS, PaDXR, PaHMGR and cytochrome P450 monooxygenase genes, PaCYP1 and PaCYP9 were highly upregulated in RB (1:1) and green. The overall results suggest RB (1:1) as the better lighting option amongst the studied spectral lights for obtaining maximum phytochemicals in P. amboinicus. Work is being continued with different spectral ratios of red and blue LED lights to maximize phytochemical accumulation, the outcome of which will be reported elsewhere in near future. Supplementary Information: The online version contains supplementary material available at 10.1007/s00344-023-11028-6.

Temporal accumulation of pigments during colour transformation from white to red in Combretum indicum (L.) DeFilipps (syn. Quisqualis indica L.) flowers.

This study focuses on the identification of major anthocyanin following its temporal accumulation in colour changing flowers of Combretum indicum (L.) DeFilipps (syn. Quisqualis indica L.). Separation and identification of pigments governing changes in floral colour were performed using HPLC-DAD. Comparison of chromatographic runs with retention time and UV-Vis spectra of authentic standards determined cyanidin 3-O-glucoside as the major anthocyanin accumulating in the petals. Acid hydrolysis of anthocyanin extracts further confirmed cyanidin as the major anthocyanidin in floral tissue. Light microscopic studies revealed gradual accumulation of pigments in the epidermal and hypodermal cell layers of petals. Antioxidant potentials of floral extracts in ethanol, methanol, water and ethyl acetate were determined by DPPH assay where methanolic extracts showed highest free-radical scavenging capacity, and petals of red stage showed maximum activity. Antioxidative potentials measured in terms of FRAP and ABTS also indicated similar results showing highest activity in the red stage.

Lipid-rich endo-metabolites from a vertically transmitted fungal endophyte Penicillium sp. PM031 attenuate virulence factors of phytopathogenic Ralstonia solanacearum.

The bacterial wilt caused by Ralstonia solanacearum is a destructive plant disease globally. Since a completely non-biological control measure could be a matter of environmental concern, investigations of developing eco-friendly strategies are required to control this phytopathogen. Attenuation of the bacterial virulence in addition to destroying the pathogen may be an alternative and overarching approach to control this disease. In this study, we have explored the potentiality of a vertically transmitted endophytic fungus Penicillium sp. PM031 isolated from stem of in vitro grown, wilt susceptible tomato cultivar to control this phytopathogen. The endophytic fungus was unable to inhibit the bacterial growth during direct confrontation in co-culture system; rather its growth and extracellular secretion were affected by the bacterium. Interestingly, the PM031-derived endo-metabolites, containing ~80% of lipid molecules, showed the dose-dependent growth inhibitory effect against R. solanacearum. Metabolite treatment with a concentration of 2500 and 5000 µg/ml significantly inhibited the bacterial growth 24.72% and 64.31%, respectively. Higher concentrations of endo-metabolite treatment exhibited antibacterial activity by rupturing cellular membranes. Furthermore, the endo-metabolites negatively influence the virulence factors necessary in early phases of bacterial infection, such as motility and biofilm formation. Our study highlights even if an endophytic fungus associated with the susceptible host plant cannot tackle R. solanacearum directly, its lipid-rich metabolites have potential to attenuate the virulence of phytopathogen. We believe this study can be a stepping stone to develop suitable formulations to control the bacterial wilt in a sustainable way, which will reduce excessive uses of synthetic bactericides.

Characterization of an alcohol acetyltransferase GcAAT responsible for the production of antifungal volatile esters in endophytic Geotrichum candidum PF005.

Alcohol acetyltransferases (AATs) are a group of enzymes that catalyze the formation of esters from different alcohols and acetyl-CoA. However, these enzymes are not well characterized with regard to synthesis of antifungal compounds. The present study aims to investigate the AAT enzyme from Geotrichum candidum PF005, an endophytic yeast-like fungus that emits fruity scented antifungal volatiles, primarily comprising of acetate esters. After PCR-based cloning of the GcAAT gene, the encoded enzyme was characterized structurally through in silico methods and functionally via heterologous expression in Saccharomyces cerevisiae. In native host, the single copy GcAAT gene exhibited induced expression upon supplementation with metabolic precursors, like L-leucine (Leu) or α-ketoisocaproate (α-KIC). Docking studies using the modelled structure of GcAAT revealed differential but favourable binding interactions for three alcohol substrates (i.e., isoamyl alcohol, isobutyl alcohol and 2-phenylethanol) and the co-substrate acetyl-CoA. Binding sites for both substrate and co-substrate are found to be located inside a tunnel identified in the structure, wherein the H208 of the acetyltransferase conserved motif HXXXD was found at a hydrogen bond distance from the substrate. Functional complementation of GcAAT in S. cerevisiae AAT knockout strain caused 32% decrease in dry biomass weight of the test phytopathogenic fungus, Rhizoctonia solani as compared to the control (AAT knockout strain with empty plasmid) after 72 h of incubation due to the emitted volatiles. When the transformed yeast cells were fed with Leu and α-KIC, the relative abundance of the isoamyl acetate ester increased by 21% and 48%, respectively as compared to the control (without precursor). Further analysis documented that volatiles from α-KIC fed GcAAT transformant exhibited 58% higher antifungal activity against the test fungus R. solani than the control, engendered by increased oxidative stress that led to distorted mycelial morphology and increased hyphal branching. Together, the augmented antifungal effect displayed by the GcAAT expressing S. cerevisiae AAT knockout strain is clearly attributable to the acetate esters, especially isoamyl acetate, which are inherently produced in endophytic G. candidum PF005 as antifungal volatiles.

An insight into fruit aroma volatilome during postharvest maturation in two popular Musa cultivars of tropics.

BACKGROUND: Banana is one of the major global horticultural fruit crops cultivated in the humid tropics and subtropics. Fruit quality and consumer acceptability of any climacteric fruit depend mainly on its postharvest aroma volatile profiles. The present study aimed to profile fruit volatiles status during postharvest storage of two banana cultivars: Kanthali (Musa sp. cv. Kanthali, Kt) and Kacha Kela (Musa sp. cv. Kacha Kela, Kk) from the ABB genome group. RESULTS: Both cultivars showed differences in the soluble sugar contents, with Kt being higher than Kk. The volatile compounds were profiled from the pulp as emitted, endogenous and glycosyl-bound forms, along with peel-endogenous and whole fruit volatiles during postharvest storage. Both cultivars showed a wide range of variations in volatile aroma pools; nevertheless, esters and aliphatic compounds were found to be the major contributors of fruit volatiles in Kt and Kk, respectively. The pulp-endogenous volatiles served as the major pool, which showed a sharp decline with a corresponding increase of emission. Many volatiles were found to be glycosylated during early postharvest storage, with de-glycosylation occurring with an increase in storage time, resulting in fruit softening and a concurrent supply of sugar bound volatiles towards emission. CONCLUSION: As a whole, the study outcome provides an overview of fruit volatilome during postharvest storage and suggests a possible inter-linking among the volatile components in the cultivars. It is plausible that the release of aroma volatiles from pulp is mediated via peel, with volatiles accumulating as peel-endogenous volatiles representing the temporary pool reservoir. © 2022 Society of Chemical Industry.

Revealing floral metabolite network in tuberose that underpins scent volatiles synthesis, storage and emission.

KEY MESSAGE: The role of central carbon metabolism in the synthesis and emission of scent volatiles in tuberose flowers was revealed through measurement of changes in transcripts and metabolites levels. Tuberose or Agave amica (Medikus) Thiede & Govaerts is a widely cultivated ornamental plant in several subtropical countries. Little is known about metabolite networking involved in biosynthesis of specialized metabolites utilizing primary metabolites. In this study, metabolite profiling and gene expression analyses were carried out from six stages of maturation throughout floral lifespan. Multivariate analysis indicated distinction between early and late maturation stages. Further, the roles of sugars viz. sucrose, glucose and fructose in synthesis, glycosylation and emission of floral scent volatiles were studied. Transcript levels of an ABC G family transporter (picked up from the floral transcriptome) was in synchronization with terpene volatiles emission during the anthesis stage. A diversion from phenylpropanoid/benzenoid to flavonoid metabolism was observed as flowers mature. Further, it was suggested that this metabolic shift could be mediated by isoforms of 4-Coumarate-CoA ligase along with Myb308 transcription factor. Maximum glycosylation of floral scent volatiles was shown to occur at the late mature stage when emission declined, facilitating both storage and export from the floral tissues. Thus, this study provides an insight into floral scent volatiles synthesis, storage and emission by measuring changes at transcripts and metabolites levels in tuberose throughout floral lifespan.

Glandular trichomes of the flowers and leaves in Millingtonia hortensis (Bignoniaceae).

MAIN CONCLUSION: Three types of the glandular trichomes are developed on the flowers and leaves of Millingtonia hortensis. Morphology, cell ultrastructure and content of the volatile compounds are specific to each trichome type. The aim of this study was to characterize the structural and histochemical features of the glandular trichomes (GTs) of two types localized on the different flower parts and leaves in Millingtonia hortensis, as well as to identify the composition of the internal pool of metabolites. The peltate GTs are most common; they are founded on peduncle, calyx, ovary, and leaves. GTs consist of 12-24-cell disk-shaped head and a single-celled neck. The capitate GTs are located on corolla tube and have four to eight-cell head, single-celled neck and a wide multicellular stalk. A series of histochemical reactions and fluorescent microscopy revealed the various substances in the chemical composition of GTs. Acid polysaccharides are predominately identified in the capitate trichomes of the corolla tube and peltate trichomes of calyx, terpenes present in larger quantity in the trichomes of the corolla tube and ovary, whilst phenolic substances prevail in the trichomes of the calyx and ovary. GTs of each type are characterized by specific ultrastructural traits. Smooth endoplasmic reticulum (SER) and leucoplasts prevail in the peltate trichomes of peduncle, calyx and ovary; Golgi apparatus is the common organelle in the capitate trichomes of the corolla tube and peltate trichomes of calyx; the huge aggregates of the RER cisterns there are in cytoplasm of all leaf trichomes. Synthesized secretion accumulates in the subcuticular cavity of all GTs except the leaf peltate trichomes. In the trichomes of the leaves secretion is stored in the thick upper cell wall with the wide cutinized layer. For the first time content of the internal pool of metabolites from the flowers and leaves was identified by GC-MS. Seventeen compounds, including alcohols, fatty acid derivatives, monoterpenes, sesquiterpenes, and benzenoids were identified. 1-octen 3-ol, 3-carene, methyl salicylate, p-hydroxybenzeneethanol and 1-hydroxy-2,4-di-tertbutyl-benzene were the main compounds of the flower scent. We consider GTs of the reproductive organs in M. hortensis synthesizing acid polysaccharides and volatile compounds as secretory structures attracting of pollinators, whereas the leaf peltate trichomes accumulating predominately non-volatile phenols, protect young vegetative shoots against small herbivorous insects and pathogens.

Specialized metabolites contributing to colour and scent volatiles in Uvaria hamiltonii flowers.

The present study focuses on the emitted and endogenous scent profiles of Uvaria hamiltonii flowers. Among the 34 compounds identified, sesquiterpenoids were found to dominate the floral volatiles composition. Profiles from endogenous scent volatiles showed higher number of compounds than the emitted ones. The anthocyanin pigment responsible for the flower colour was also explored. It was found that a single anthocyanin compound, cyanidin-3-O-glucoside, was principally responsible for petal colour. Total phenolic content was evaluated and antioxidant capacities were studied with the help of DPPH, FRAP and ABTS assays. The total phenolic content and the antioxidant capacity were higher in methanolic extract as compared to aqueous, petroleum ether and ethyl acetate extracts of U. hamiltonii flowers.

Impact of seed-transmitted endophytic bacteria on intra- and inter-cultivar plant growth promotion modulated by certain sets of metabolites in rice crop.

Exploring the beneficial interactions between plant and endophytes could be an effective strategy in the implementation of sustainable agricultural practices to enhance crop productivity. In this study, we aimed to evaluate holistically the plant growth promoting (PGP) abilities rendered by seed-transmitted endophytic bacteria isolated from in vitro grown calli of two rice cultivars. Nine bacterial endophytes, designated as PB001-PB009, were isolated and identified at the genus level through 16S rRNA gene sequence analysis. Biochemical investigations disclosed that they possess several PGP traits, such as phosphate solubilization, indole acetic acid biosynthesis, ammonia production, nitrogen fixation, amylase production and siderophore production. Results in gnotobiotic conditions revealed an increase in fresh weight, dry weight, root length and shoot length of seedlings germinated from endophyte-primed seeds than the control (uninoculated) set in a non-host and two host rice cultivars. In net house experiments, plants germinated from Micrococcus sp. PB001, Pseudomonas sp. PB002, Methylobacterium sp. PB005 and Methylorubrum sp. PB009 primed seeds showed an increase of upto 34.06 %, 38.77 %, 182.87 %, 16.59 % and 33.52 % in chlorophyll content, number of tillers/plant, number of grains/plant, grain size and grain weight, respectively than control plant sets in the non-host rice cultivar, further validating inter-cultivar PGP abilities of these endophytes. Metabolite profiling unfolded the abundance of few metabolites that are involved in pathways associated with PGP traits, in seedlings germinated from the endophyte-primed seeds. Together, the study documents the effect of seed-transmitted endophytic bacteria on intra- and inter-cultivar PGP by modulating certain sets of metabolites in rice plant, and is promising in developing bioinoculant formulations employing these selected endophytes for enhancement of rice productivity.

Replacing critical point drying with a low-cost chemical drying provides comparable surface image quality of glandular trichomes from leaves of Millingtonia hortensis L. f. in scanning electron micrograph.

Sample preparation including dehydration and drying of samples is the most intricate part of scanning electron microscopy. Most current sample preparation protocols use critical-point drying with liquid carbon dioxide. Very few studies have reported samples that were dried using chemical reagents. In this study, we used hexamethyldisilazane, a chemical drying reagent, to prepare plant samples. As glandular trichomes are among the most fragile and sensitive surface structures found on plants, we used Millingtonia hortensis leaf samples as our study materials because they contain abundant glandular trichomes. The results obtained using this new method are identical to those produced via critical-point drying.

Profiling of volatile and non-volatile metabolites in Polianthes tuberosa L. flowers reveals intraspecific variation among cultivars.

Polianthes tuberosa L. (tuberose) is a widely cultivated ornamental crop in Asian countries. Different cultivars of tuberose have been developed through breeding programs in India. However, no reports on floral fragrance and metabolite contents of these cultivars are available. In this study, an attempt has been made to evaluate the levels of both volatile and non-volatile metabolites from seven different cultivars of P. tuberosa. Presence of benzenoids, phenylpropanoids, terpenoids, and few fatty acid derivatives as emitted, endogenous and glycosylated forms were revealed from the studied cultivars. Further, chemometric analyses in both supervised and unsupervised manner led to identification of patterns among the cultivars. Among the seven cultivars, four distinct clusters were obtained linking to their volatiles, flavonoids and primary metabolite levels. Metabolic variations obtained from the cultivars also suggest cross-talks between phenylpropanoid, benzenoid, and flavonoid pathways. Thus metabolite profiling reported here may help in characterization of tuberose cultivars for perfumery utility and future breeding programme.

Developmental variation in floral volatiles composition of a fragrant orchid Zygopetalum maculatum (Kunth) Garay.

Emitted scent volatile profile of an orchid species Zygopetalum maculatum was studied using dynamic headspace sampling technique with four different adsorbent matrices, namely Porapak Type Q polymer (mesh size: 80/100), Tenax (mesh size: 60/80), activated charcoal and graphite. In addition, developmental variations in scent emissions and endogenous volatile levels were also investigated. Gas chromatography-mass spectrometry analysis revealed the presence of 21 volatile compounds in the headspace, which was predominantly enriched with benzenoid compounds. Among these benzenoids, o-diethylbenzene and p-diethylbenzene were the major compounds followed by benzyl acetate and methyl salicylate. Among the phenylpropanoid compounds, 2-phenylethyl acetate was the major volatile. However, as compared to benzenoids, the quantity was much lesser, indicating the inclination of phenylalanine flux towards benzenoid pathway. The outcome of this study has the implications in enhancing fragrance and vase life of orchids of the Sikkim Himalaya region and thus may further help to meet the growing market demand.

Temporal relationship between emitted and endogenous floral scent volatiles in summer- and winter-blooming Jasminum species.

Jasminum spp. is cultivated for their fragrant flowers used in essential oil production and cosmetic uses. An attempt was made to study the temporal variations in floral scent volatiles composition including emitted, free endogenous and glycosyl-linked volatile compounds from two summer-blooming species namely, Jasminum auriculatum and Jasminum grandiflorum as well as from two winter-blooming species namely, Jasminum multiflorum and Jasminum malabaricum. The overall emitted volatile organic compounds (VOCs) were found to be highest when the matrix Porapak Q 80/100 was used with dichloromethane (DCM) as elution solvent. The floral volatile emission from bud to senescence exhibited nocturnal maxima pattern for both the summer-blooming species. Both the winter-blooming species emitted its highest concentration at noon. The free endogenous concentrations of all VOCs were low when corresponding emitted concentrations were high. Enzymatic treatment of petal extract revealed that several aromatic volatiles including aromatic alcohols and monoterpenols are synthesized and stored in the flowers as water-soluble glycosides; these compounds were shown to accumulate in higher amounts in flowers at late bud stage. These findings indicate the utilization of the precursors, i.e. the volatile-conjugates, through hydrolysis followed by their release as free-volatiles at flower opening stage. The outcome as a whole suggests a linkage among the temporal pattern of emitted volatiles, free-endogenous volatiles and glycoside-bound volatile compounds in all above studied Jasminum spp. and provided an overview of their floral volatilome.

Quorum sensing inhibitory activity of the metabolome from endophytic Kwoniella sp. PY016: characterization and hybrid model-based optimization.

Quorum sensing, the microbial communication system, is gaining importance as a therapeutic target against pathogens. The two key reasons for the rising demand of quorum sensing (QS) inhibitory molecules are low selective pressure to develop resistance by pathogens and possibility of more species-specific effects. Due to complex interactions in a unique niche of live plant tissues, endophytes, as a survival mechanism, potentially produce various bioactive compounds such as QS inhibitors. We report the isolation of an endophytic fungus Kwoniella sp. PY016 from the medicinal plant "Bahera" (Terminalia bellirica), which exhibits substantial quorum sensing inhibition and anti-biofilm activities against the standard test organism, Chromobacterium violaceum. Sugar, sugar alcohol, carboxylic acid, lipid, and phenolic classes of metabolites (predominantly xylitol) are responsible components of the metabolome for the desired bioactivity. A judicious combination of single-factor-at-a-time strategy and artificial neural network modeling combined with genetic algorithm was employed for the selection and optimization of the critical process and medium parameters. Through this newly adopted hybrid model-based optimization, the quorum sensing inhibitory activity of the endophytic metabolome was increased by ~ 30%. This is the first report on optimization of QS inhibitory activity from any fungal endophyte using such a hybrid advanced approach.

Characterization and Synergistic Effect of Antifungal Volatile Organic Compounds Emitted by the Geotrichum candidum PF005, an Endophytic Fungus from the Eggplant.

Plant-associated endophytes are recognized as sources of novel bioactive molecules having diverse applications. In this study, an endophytic yeast-like fungal strain was isolated from the fruit of eggplant (Solanum melongena) and identified as Geotrichum candidum through phenotypic and genotypic characterizations. This endophytic G. candidum isolate PF005 was found to emit fruity scented volatiles. The compositional profiling of volatile organic compounds (VOCs) revealed the presence of 3-methyl-1-butanol, ethyl 3-methylbutanoate, 2-phenylethanol, isopentyl acetate, naphthalene, and isobutyl acetate in significant proportion when analyzed on a time-course basis. The VOCs from G. candidum exhibited significant mycelial growth inhibition (54%) of phytopathogen Rhizoctonia solani, besides having mild antifungal activity against a few other fungi. The source of carbon as a nutrient was found to be an important factor for the enhanced biosynthesis of antifungal VOCs. The antifungal activity against phytopathogen R. solani was improved up to 91% by feeding the G. candidum with selective precursors of alcohol and ester volatiles. Furthermore, the antifungal activity of VOCs was enhanced synergistically up to 92% upon the exogenous addition of naphthalene (1.0 mg/plate). This is the first report of G. candidum as an endophyte emitting antifungal VOCs, wherein 2-penylethanol, isopentyl acetate, and naphthalene were identified as important contributors to its antifungal activity. Possible utilization of G. candidum PF005 as a mycofumigant has been discussed based upon its antifungal activity and the qualified presumption of safety status.

Morphological, Physiological and Ultrastructural Changes in Flowers Explain the Spatio-Temporal Emission of Scent Volatiles in Polianthes tuberosa L.

Tuberose or Polianthes tuberosa L. is a horticultural crop of tropical origin, widely cultivated for its pleasant and intense floral fragrance in the evening. Here an investigation was made into the physiological and cell biological aspects of floral scent biosynthesis, tissue localization and emission that have not previously been examined. Volatiles collected from floral headspace were analyzed by gas chromatography-mass spectrometry (GC-MS) for identification of individual compounds and elucidation of emission patterns. Transcript accumulation and the amount of active enzyme were measured to understand the enzymatic route of scent volatile biosynthesis. Localization of scent volatiles was investigated by histochemical and ultrastructural studies. Scent emission was found to be rhythmic and nocturnal under normal day-night influence, peaking at night. Enhanced enzyme activities and transcript accumulation were recorded just prior to maximum emission. Through scanning electron microscopy (SEM) analysis, the presence of a large number of floral stomata on the adaxial surface of the tepal was revealed which might have bearing on tissue-specific emission. Guard cells of stomata responded significantly to histochemical tests, which also indicated that epidermal tissues are mostly involved in scent emission. High metabolic activity was found in epidermal layers during anthesis as shown by transmission electron microscopy (TEM) analysis. Further, new insight into the localization of scent compounds, the plausible tissue involved in their release along with the preceding ultrastructural changes at the cellular levels is presented. Finally, ultrastructural analysis of the tepal surface has been able to fill a major gap in knowledge of stomatal involvement during scent emission.

Enzymatic production and emission of floral scent volatiles in Jasminum sambac.

Floral scent composed of low molecular weight volatile organic compounds. The sweet fragrance of any evening blooming flower is dominated by benzenoid and terpenoid volatile compounds. Floral scent of Jasminum sambac (Oleaceae) includes three major benzenoid esters - benzylacetate, methylbenzoate, and methylsalicylate and three major terpene compounds viz. (E)-ß-ocimene, linalool and α-farnesene. We analyzed concentrations and emission rates of benzenoids and terpenoids during the developmental stages of J. sambac flower. In addition to spatial emission from different floral parts, we studied the time-course mRNA accumulations of phenylalanine ammonia-lyase (PAL) and the two representative genes of terpenoid pathway, namely 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) and terpene synthase (TPS). Further, in vitro activities of several enzymes of phenylpropanoid/benzenoid pathway viz., PAL and acetyl-coenzyme A: benzylalcohol acetyltransferase (BEAT), S-adenosyl-l-methionine: benzoic acid carboxyl methyl transferase (BAMT) and S-adenosyl-l-methionine: salicylic acid carboxyl methyltransferase (SAMT) were studied. All the above enzyme activities along with the in vitro activities of DXR and TPS were found to follow a certain rhythm as observed in the emission of different benzenoid and terpenoid compounds. Linalool emission peaked after petal opening and coincided with maximal expression of JsTPS gene as evidenced from RT-PCR analyses (semi-quantitative). The maximum transcript accumulation of this gene was observed in flower petals, indicating that the petals of J. sambac flower play an important role as a major contributor of volatile precursors. The transcripts accumulation of JsDXR and JsTPS in different developmental stages and in different floral part showed that emissions of terpenoid volatiles in J. sambac flower are partially regulated at transcription levels.

Biostimulation of Indigenous Microbial Community for Bioremediation of Petroleum Refinery Sludge.

Nutrient deficiency severely impairs the catabolic activity of indigenous microorganisms in hydrocarbon rich environments (HREs) and limits the rate of intrinsic bioremediation. The present study aimed to characterize the microbial community in refinery waste and evaluate the scope for biostimulation based in situ bioremediation. Samples recovered from the wastewater lagoon of Guwahati refinery revealed a hydrocarbon enriched [high total petroleum hydrocarbon (TPH)], oxygen-, moisture-limited, reducing environment. Intrinsic biodegradation ability of the indigenous microorganisms was enhanced significantly (>80% reduction in TPH by 90 days) with nitrate amendment. Preferred utilization of both higher- (>C30) and middle- chain (C20-30) length hydrocarbons were evident from GC-MS analysis. Denaturing gradient gel electrophoresis and community level physiological profiling analyses indicated distinct shift in community's composition and metabolic abilities following nitrogen (N) amendment. High throughput deep sequencing of 16S rRNA gene showed that the native community was mainly composed of hydrocarbon degrading, syntrophic, methanogenic, nitrate/iron/sulfur reducing facultative anaerobic bacteria and archaebacteria, affiliated to γ- and δ-Proteobacteria and Euryarchaeota respectively. Genes for aerobic and anaerobic alkane metabolism (alkB and bssA), methanogenesis (mcrA), denitrification (nirS and narG) and N2 fixation (nifH) were detected. Concomitant to hydrocarbon degradation, lowering of dissolve O2 and increase in oxidation-reduction potential (ORP) marked with an enrichment of N2 fixing, nitrate reducing aerobic/facultative anaerobic members [e.g., Azovibrio, Pseudoxanthomonas and Comamonadaceae members] was evident in N amended microcosm. This study highlighted that indigenous community of refinery sludge was intrinsically diverse, yet appreciable rate of in situ bioremediation could be achieved by supplying adequate N sources.