Pectin Methylesterase of Datura species,purification, and characterization from Datura stramoniumand 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.     

鳞杯伞α-半乳糖苷酶的提取纯化及酶学性质研究

采用离子交换层析和凝胶过滤层析对鳞杯伞子实体中的α-半乳糖苷酶进行纯化,得到了一种分子量为50 kDa的α-半乳糖苷酶,命名为CSG。纯化后的CSG纯化倍数为891.46倍,比活力为54.78 U/mg,得率为0.71%。通过BLAST比对液相色谱-串联质谱(LC-MS/MS)获得其肽段,发现其为GH27家族的α-半乳糖苷酶。CSG的最适pH为3.0,最适温度为50 ℃。在酸性范围pH 2.2-7.0和温度范围4-30 ℃有较好的稳定性。Mn²⁺、Cd²⁺、Cu²⁺对CSG有较强的抑制作用。半乳糖和蜜二糖对CSG的抑制类型为混合型抑制。化学修饰剂N-溴代琥珀酰亚胺显著降低CSG的活力,碳二亚胺对CSG具有显著的激活作用。该酶具有良好的蛋白酶抗性,且对棉子糖家族寡糖(RFOs)、瓜尔豆胶和赤槐豆胶均表现出良好的水解作用。     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Germination responses to temperature and water potential in Jatropha curcas seeds: a hydrotime model explains the difference between dormancy expression and dormancy induction at different incubation temperatures

Background and Aims

Jatropha curcas is a drought-resistant tree whose seeds are a good source of oil that can be used for producing biodiesel. A successful crop establishment depends on a rapid and uniform germination of the seed. In this work we aimed to characterize the responses of J. curcas seeds to temperature and water availability, using thermal time and hydrotime analysis,

Methods

Thermal and hydrotime analysis was performed on germination data obtained from the incubation of seeds at different temperatures and at different water potentials.

Key Results

Base and optimum temperatures were 14·4 and 30 °C, respectively. Approximately 20 % of the seed population displayed absolute dormancy and part of it displayed relative dormancy which was progressively expressed in further fractions when incubation temperatures departed from 25 °C. The thermal time model, but not the hydrotime model, failed to describe adequately final germination percentages at temperatures other than 25 °C. The hydrotime constant, θ_H, was reduced when the incubation temperature was increased up to 30 °C, the base water potential for 50 % germination,Ψ_b(50), was less negative at 20 and 30 °C than at 25 °C, indicating either expression or induction of dormancy. At 20 °C this less negative Ψ_b(50) explained satisfactorily the germination curves obtained at all water potentials, while at 30 °C it had to be corrected towards even less negative values to match observed curves at water potentials below 0. Hence, Ψ_b(50) appeared to have been further displaced to less negative values as exposure to 30 °C was prolonged by osmoticum. These results suggest expression of dormancy at 20 °C and induction of secondary dormancy above 25 °C. This was confirmed by an experiment showing that inhibition of germination imposed by temperatures higher than 30 °C, but not that imposed at 20 °C, is a permanent effect.

Conclusions

This study revealed (a) the extremely narrow thermal range within which dormancy problems (either through expression or induction of dormancy) may not be encountered; and (b) the high sensitivity displayed by these seeds to water shortage. In addition, this work is the first one in which temperature effects on dormancy expression could be discriminated from those on dormancy induction using a hydrotime analysis.     

Effects of the Mi-1, N and Tabasco Genes on Infection and Reproduction of Meloidogyne mayaguensis on Tomato and Pepper Genotypes

Meloidogyne mayaguensis is a damaging root-knot nematode able to reproduce on root-knot nematode-resistant tomato and other economically important crops. In a growth chamber experiment conducted at 22 and 33°C, isolate 1 of M. mayaguensis reproduced at both temperatures on the Mi-1-carrying tomato lines BHN 543 and BHN 585, whereas M. incognita race 4 failed to reproduce at 22°C, but reproduced well at 33°C. These results were confirmed in another experiment at 26 ± 1.8°C, where minimal or no reproduction of M. incognita race 4 was observed on the Mi-1-carrying tomato genotypes BHN 543, BHN 585, BHN 586 and ‘Sanibel’, whereas heavy infection and reproduction of M. mayaguensis isolate 1 occurred on these four genotypes. Seven additional Florida M. mayaguensis isolates also reproduced on resistant ‘Sanibel’ tomato at 26 ± 1.8°C. Isolate 3 was the most virulent, with reproduction factor (Rf) equal to 8.4, and isolate 8 was the least virulent (Rf = 2.1). At 24°C, isolate 1 of M. mayaguensis also reproduced well (Rf ≥ 1) and induced numerous small galls and large egg masses on the roots of root-knot nematode-resistant bell pepper ‘Charleston Belle’ carrying the N gene and on three root-knot nematode-resistant sweet pepper lines (9913/2, SAIS 97.9001 and SAIS 97.9008) carrying the Tabasco gene. In contrast, M. incognita race 4 failed to reproduce or reproduced poorly on these resistant pepper genotypes. The ability of M. mayaguensis isolates to overcome the resistance of tomato and pepper genotypes carrying the Mi-1, N and Tabasco genes limits the use of resistant cultivars to manage this nematode species in infested tomato and pepper fields in Florida.     

Establishment of a leukocyte cell line derived from peritoneal macrophages of fish,Labeo rohita (Hamilton, 1822)

A continuous leukocyte cell line with phagocytic activity was established from peritoneal macrophages of rohu, Labeo rohita (LRPM). LRPM was initiated from adherent mononuclear leukocytes isolated from peritoneal cavity of rohu, without use of any growth factors or feeder cells. These cells exhibited maximum growth at 30 °C in L-15 medium containing 20 % foetal bovine serum, and has been subcultured for more than 60 passages till date. The cells showed 85 % viability after 6 months of storage in liquid nitrogen. The species of origin of the LRPM was confirmed by the amplification and sequencing of 655 bp fragment of cytochrome oxidase subunit I of mitochondrial DNA. Functionally, LRPM showed phagocytic activity of yeast cells and fluorescent latex beads as evaluated by phase contrast and scanning electron microscopy, respectively. Immuno-modulators such as bacterial lipopolysaccharide and phorbol myristate acetate resulted in functional activation of LRPM; and enhanced their microbicidal activity through release of reactive oxygen species and nitric oxide. Culture supernatant from activated cells also revealed lysozyme activity. Cells of LRPM were positive for alpha-naphthyl acetate esterase enzyme indicating macrophage lineage. Our results indicate that this cell line can be a useful in vitro tool to study the role of macrophages in teleost immune system and to evaluate the effects of new aquaculture drugs. The LRPM cell line represents the first reported leukocyte cell line of peritoneal origin from any freshwater species of fish.     

Thermostable Xanthine Oxidase Activity from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RL-2d Isolated from Manikaran Thermal Spring: Production and Characterization

Xanthine oxidase is an important enzyme of purine metabolism that catalyzes the hydroxylation of hypoxanthine to xanthine and then xanthine to uric acid. A thermostable xanthine oxidase is being reported from a thermophilic organism RL-2d isolated from the Manikaran (Kullu) hot spring of Himachal Pradesh (India). Based on the morphology, physiological tests, and 16S rDNA gene sequence, RL-2d was identified as Bacillus pumilus. Optimization of physiochemical parameters resulted into 4.1-fold increase in the xanthine oxidase activity from 0.051 U/mg dcw (dry cell weight) to 0.209 U/mg dcw. The xanthine oxidase of B. pumilus RL-2d has exhibited very good thermostability and its t_1/2 at 70 and 80 °C were 5 and 1 h, respectively. Activity of this enzyme was strongly inhibited by Hg²⁺, Ag⁺ and allopurinol. The investigation showed that B. pumilus RL-2d exhibited highest xanthine oxidase activity and remarkable thermostability among the other xanthine oxidases reported so far.     

Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated from Solanum torvum Swartz. Fruit

Abstract

Solanum torvum Swartz. (Solanaceae) fruit is traditionally used for the treatment of bacterial and fungal infections. The methanolic extract was subjected to activity guided fractionation by column chromatography over silica gel. The structure of the compound was elucidated using physical and spectroscopic data. The antimicrobial activity was screened using five Gram-positive bacteria, six Gram-negative bacteria, seven clinical isolates and four fungi. Antimycobacterial activity was screened against two Mycobacterium strains. The zone of inhibition by methyl caffeate ranged from 0 to 22 mm. The lowest minimum inhibitory concentration (MIC) values of methyl caffeate were: 50 μg/ml against P. vulgaris, 25 μg/ml against K. pneumoniae (ESBL-3971), 8 μg/ml against M. tuberculosis (H³⁷Rv) and 8 μg/ml against M. tuberculosis (Rif^R). Methyl caffeate showed moderate antimicrobial and prominent antimycobacterial activities. Methyl caffeate can be evaluated further for drug development.     

Efficient Production of Lactic Acid from Sweet Sorghum Juice by a Newly Isolated Lactobacillus salivarius CGMCC 7.75

Sweet sorghum juice was a cheap and renewable resource, and also a potential carbon source for the fermentation production of lactic acid (LA) by a lactic acid bacterium. One newly isolated strain Lactobacillus salivarius CGMCC 7.75 showed the ability to produce the highest yield and optical purity of LA from sweet sorghum juice. Studies of feeding different concentrations of sweet sorghum juice and nitrogen source suggested the optimal concentrations of fermentation were 325 ml l⁻¹ and 20 g l⁻¹, respectively. This combination produced 142.49 g l⁻¹ LA with a productivity level of 0.90 g of LA per gram of sugars consumed. The results indicated the high LA concentration achieved using L. salivarius CGMCC 7.75 not only gives cheap industrial product, but also broaden the application of sweet sorghum.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-013-0377-0) contains supplementary material, which is available to authorized users.     

Variation in the functioning of autonomous self-pollination, pollinator services and floral traits in three Centaurium species

Background and Aims

Reproductive assurance through autonomous selfing is thought to be one of the main advantages of self-fertilization in plants. Floral mechanisms that ensure autonomous seed set are therefore more likely to occur in species that grow in habitats where pollination is scarce and/or unpredictable.

Methods

Emasculation and pollen supplementation experiments were conducted under laboratory conditions to investigate the capacity for, and timing of autonomous selfing in three closely related Centaurium species (Centaurium erythraea, C. littorale and C. pulchellum). In addition, observations of flower visitors were combined with emasculation and pollen addition experiments in natural populations to investigate the degree of pollinator limitation and pollination failure and to assess the extent to which autonomous selfing conferred reproductive assurance.

Results

All three species were capable of autonomous selfing, although this capacity differed significantly between species (index of autonomous selfing 0·55 ± 0·06, 0·68 ± 0·09 and 0·92 ± 0·03 for C. erythraea, C. littorale and C. pulchellum, respectively). The efficiency and timing of autogamous selfing was primarily associated with differences in the degree of herkogamy and dichogamy. The number of floral visitors showed significant interspecific differences, with 1·6 ± 0·6, 5·4 ± 0·6 and 14·5 ± 2·1 floral visitors within a 2 × 2 m² plot per 20-min observation period, for C. pulchellum, C. littorale and C. erythraea, respectively. Concomitantly, pollinator failure was highest in C. pulchellum and lowest in C. erythraea. Nonetheless, all three study species showed very low levels of pollen limitation (index of pollen limitation 0·14 ± 0·03, 0·11 ± 0·03 and 0·09 ± 0·02 for C. erythraea, C. littorale and C. pulchellum, respectively), indicating that autonomous selfing may guarantee reproductive assurance.

Conclusions

These findings show that limited availability of pollinators may select for floral traits and plant mating strategies that lead to a system of reproductive assurance via autonomous selfing.     

Optimization of Antimicrobial Production by a Marine Actinomycete Streptomyces afghaniensis VPTS3-1 Isolated from Palk Strait, East Coast of India

Totally 25 marine soil samples were collected from the region of Palk Strait of Bay of Bengal, Tamil Nadu, and were subjected to the isolation of actinomycetes. Sixty-eight morphologically distinct isolates were obtained and 37% (25) of them had antimicrobial activity. The potential producer was named as Streptomyces sp. VPTS3-1 and the phylogenetic evaluation on the basis of 16S rDNA sequence further categorized the organism as Streptomyces afghaniensis VPTS3-1. Further, the antimicrobial compound was extracted from the isolate using various solvents and the antimicrobial efficacies were tested against bacterial and fungal pathogens. In addition, in vitro optimization of parameters for the antimicrobial compound production revealed that the suitable pH as 7–8, the period of incubation as 9 days, temperature (30°C), salinity (2%), and starch and KNO₃ as the suitable carbon and nitrogen sources respectively in starch–casein medium.     

Morphological and Molecular Characterization of Pomegranate Fruit Rot Pathogen, <Emphasis Type="Italic">Chaetomella raphigera</Emphasis>, and its Virulence Factors

A new fungal pathogen was isolated from rotten pomegranates collected from the orchards of different parts of Maharashtra. The pathogen was morphologically identified as Chaetomella raphigera followed by sequencing of ITS and D1/D2 hypervariable region of LSU (28S) of rRNA gene. The pathogen produced pectinase, cellulase, xylanase and protease in liquid medium at a concentration of 71, 13.8, 54.3 and 7 U/ml respectively. Enzyme activity was also determined during pathogenesis in the tissues artificially infected by C. raphigera. Xylanase activity was maximum (25.1 U/g) followed by pectinase (19.2 U/g) and cellulase (1.5 U/g), whereas, protease activity was unnoticed. There was significant correlation (P < 0.05) between disease rating scale and pectinase, xylanase and cellulase activity in infected tissues. This indicates the simultaneous production of hydrolytic enzymes that aids in necrosis of fruit tissues. The elevated levels of these enzymes in infected tissues as compared with control suggest their possible role in pathogenesis. Thus, pectinase, cellulase and xylanase produced by C. raphigera acts as major virulence factors in the development of fruit rot in pomegranates. This is a first report of fungal fruit rot caused by C. raphigera in pomegranate.     

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

In vitro antagonism of cotton seedlings fungi and characterization of chitinase isozyme activities in Trichoderma harzianum

The antagonistic fungus Trichoderma harzianum is widely recognized as a potential biocontrol agent against several soil-borne plant pathogens. T. harzinum is rich source of chitinoltic enzymes. In vitro screening of 5 isolates of T. harzinum, one isolate of Chaetomium globosum and one isolate of Conetherium mentance, revealed that all of them had reduced growth area of Macrophomina phaseolina, Fusarium solaniand Rhizoctonia solani on PDA medium, significantly. The inhibition percentage ranged from 77.9 % to 55.9% for M. phaseolina and 59.2% to 40.4% for R. solani by T. harzinum and C. mentance, respectively. Inhibition for F. solani ranged from 76.5% to 55.7% by T. harzinum and C. globosum, respectively. Isozyme gel electrophoresis was used to assess chitinase activity secreted by selected isolates of T. harzinum under different pH degrees and temperatures. Obtained results indicated that activity of chitinase isozyme produced at 30 °C was higher than 15–20 °C for all tested isolates and activity of chitinase produced by isolates No. 4 and 5 of T. harzinum at pH (7–7.5) was higher than at pH 6, respectively.     

Isolation, Purification, and Some Properties of Penicillium chrysogenum Tannase

Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40°C. The enzyme was stable up to 30°C and within the pH range of 4.0 to 6.5. The K_m value was found to be 0.48 × 10⁻⁴ M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels.     

Purification and Characterization of a Cold-Adapted Lipase from Oceanobacillus Strain PT-11

We isolated a moderately halophilic lipase-producing bacterium from the saline soil. Based on the morphological, physiological, chemotaxonomic and phylogenetic analysis, the isolate PT-11 was postulated to be a novel species identified as Oceanobacillus rekensis PT-11. The lipase was purified 2.50-fold by Q-Sepharose FF and SP-Sepharose FF chromatography and its molecular mass was estimated to be 23.5 kDa by SDS-PAGE. It was highly active over the broad temperature ranging from 10 to 35°C and showed up to 80% of the maximum activity at 10°C indicating the lipase to be a typical cold-adapted enzyme. The enzyme activity was slightly enhanced by Na⁺, Li⁺ and K⁺. Incubation with detergents, such as Tween-20 and Tween-80, slightly inhibited the enzyme activity; while Triton X-100decreased the enzyme activity. The enzyme was fairly stable in the presence of long-chain alcohols but was highly denatured in hydrophilic solvents such as acetone or short-chain alcohols (C1–C3).     

De-hairing protease production by an isolated Bacillus cereus strain AT under solid-state fermentation using cow dung: Biosynthesis and properties

Agro-industrial residues and cow dung were used as the substrate for the production of alkaline protease by Bacillus cereus strain AT. The bacterial strain Bacillus cereus strain AT produced a high level of protease using cow dung substrate (4813 ± 62 U g⁻¹). Physiological fermentation factors such as the incubation time (72 h), the pH (9), the moisture content (120%), and the inoculum level (6%) played a vital role in the enzyme bioprocess. The enzyme production improved with the supplementation of maltose and yeast extract as carbon and nitrogen sources, respectively. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymogram analysis of the purified protease indicated an estimated molecular mass of 46 kDa. The protease enzyme was stable over a temperature range of 40–50 °C and pH 6–9, with maximum activity at 50 °C and pH 8. Among the divalent ions tested, Ca²⁺, Na⁺ and Mg²⁺ showed activities of 107 ± 0.7%, 103.5 ± 1.3%, and 104.6 ± 0.9, respectively. The enzyme showed stability in the presence of surfactants such as sodium dodecyl sulfate and on various commercially available detergents. The crude enzyme effectively de-haired goat hides within 18 h of incubation at 30 °C. The enzymatic properties of this protease suggest its suitable application as an additive in detergent formulation and also in leather processing. Based on the laboratory results, the use of cow dung for producing and extracting enzyme is not cumbersome and is easy to scale up. Considering its cheap cost and availability, cow dung is an ideal substrate for enzyme bioprocess in an industrial point of view.     

Partial Purification and Characterization of a Thermostable Actinomycete β-Amylase

A thermostable amylase, possibly a β-amylase from Thermoactinomyces sp. no. 2 isolated from soil, is reported. The enzyme was purified 36-fold by acetone precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration, and the molecular weight was estimated at 31,600. The enzyme was characterized by demonstration of optimum activity at 60°C and pH 7 and by retention of 70% activity at 70°C (30 min). It was stimulated by Mn²⁺ and Fe²⁺ but strongly inhibited by Hg²⁺. Maltose was the only detectable product of hydrolysis of starches and was quantitatively highest in plantain starch hydrolysate.     

Superoxide dismutase and ascorbate peroxidase are constitutively more thermotolerant than other antioxidant enzymes in Chenopodium album

Thermal stability of antioxidant defense enzymes was investigated in leaf and inflorescence of heat adaptive weed Chenopodium album. Leaf samples were taken at early and late seedling stage in December (LD, 20 °C/4 °C) and March (LM, 31 °C/14 °C). Young inflorescence (INF) was sampled at flowering in April (40 °C/21 °C). LD, LM and INF crude protein extracts were subjected to elevated temperatures (5 to 100 °C) for 30′. Superoxide dismutase (SOD) was the most heat stable enzyme followed by Ascorbate peroxidase (APX). Two heat stable SOD isozymes were visible on native-PAGE at 100 °C in both leaf and INF. Some heat stable APX isozymes were more abundant in INF than leaf. Thermostability of catalase (CAT) increased with age and increasing ambient temperatures in leaves. CAT activity was observed up to 60 °C in leaves and INF while peroxidase (POX) retained activity up to 100 °C in INF due to one thermostable isozyme. Glutathione reductase (GR), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR) showed activity up to 70 °C in both leaves and INF. DHAR activity was stable up to 60 °C while GR and MDHAR declined sharply after 40 °C. Constitutive heat stable isozymes of SOD and APX in leaves and INF may contribute towards heat tolerance in C. album.     

Functional aspects of floral nectar secretion of Ananas ananassoides, an ornithophilous bromeliad from the Brazilian savanna

Background and Aims

Several members of Bromeliaceae show adaptations for hummingbird pollination in the Neotropics; however, the relationships between floral structure, nectar production, pollination and pollinators are poorly understood. The main goal of this study was to analyse the functional aspects of nectar secretion related to interaction with pollinators by evaluating floral biology, cellular and sub-cellular anatomy of the septal nectary and nectar composition of Ananas ananassoides, including an experimental approach to nectar dynamics.

Methods

Observations on floral anthesis and visitors were conducted in a population of A. ananassoides in the Brazilian savanna. Nectary samples were processed using standard methods for light and transmission electron microscopy. The main metabolites in nectary tissue were detected via histochemistry. Sugar composition was analysed by high-performance liquid chromatography (HPLC). The accumulated nectar was determined from bagged flowers (‘unvisited’), and floral response to repeated nectar removal was evaluated in an experimental design simulating multiple visits by pollinators to the same flowers (‘visited’) over the course of anthesis.

Key Results

The hummingbirds Hylocharis chrysura and Thalurania glaucopis were the most frequent pollinators. The interlocular septal nectary, composed of three lenticular canals, extends from the ovary base to the style base. It consists of a secretory epithelium and nectary parenchyma rich in starch grains, which are hydrolysed during nectar secretion. The median volume of nectar in recently opened ‘unvisited’ flowers was 27·0 µL, with a mean (sucrose-dominated) sugar concentration of 30·5 %. Anthesis lasts approx. 11 h, and nectar secretion begins before sunrise. In ‘visited’ flowers (experimentally emptied every hour) the nectar total production per flower was significantly higher than in the ‘unvisited’ flowers (control) in terms of volume (t = 4·94, P = 0·0001) and mass of sugar (t = 2·95, P = 0·007), and the concentration was significantly lower (t = 8·04, P = 0·0001).

Conclusions

The data suggest that the total production of floral nectar in A. ananassoides is linked to the pollinators'' activity and that the rapid renewal of nectar is related to the nectary morphological features.