Comparison of polyphenol oxidase expression in glandular trichomes of solanum and lycopersicon species

Tetralobulate glandular trichomes are present on the foliage of many solanaceous species. Resistance of many of these species to insects is conditioned by the ability of trichomes to rupture upon contact and to rapidly polymerize their contents, resulting in entrapment of insects in hardened trichome exudate. In the wild potato, Solanum berthaultii, polymerization of trichome exudate is initiated by a soluble M_r 59,000 polyphenol oxidase (PPO), which is a dominant protein constituent of the organ. PPOs, although ubiquitous in angiosperms, typically display great heterogeneity in molecular weight and are found at low levels in plant cells. Because of the unusually high accumulation and tissue-specific expression of the M_r 59,000 PPO in S. berthaultii glandular trichomes, we analyzed trichome proteins of a number of Lycopersicon and Solanum species to assess the extent to which possession of the M_r 59,000 PPO is conserved. Trichomes were collected manually and examined for PPO activity, immuno-cross-reactivity with S. berthaultiiM_r 59,000 PPO, and protein content. In addition, N-terminal amino acid sequences were obtained for five trichome PPOs. All species analyzed possessed trichome PPOs similar in structure and level of expression to that of S. berthaultii. The relationship between sequences and structures of these conserved PPOs and the variable PPOs of leaf is discussed.     

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO₂ or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP⁺ is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O₂ to H₂O₂. Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.     

Branched Chain Amino Acid Metabolism in the Biosynthesis of Lycopersicon pennellii Glucose Esters

Lycopersicon pennellii Corr. (D'Arcy) an insect-resistant, wild tomato possesses high densities of glandular trichomes which exude a mixture of 2,3,4-tri-O-acylated glucose esters that function as a physical impediment and feeding deterrent to small arthropod pests. The acyl moieties are branched C₄ and C₅ acids, and branched and straight chain C₁₀, C₁₁, and C₁₂ acids. The structure of the branched acyl constituents suggests that the branched chain amino acid biosynthetic pathway participates in their biosynthesis. [¹⁴C]Valine and deuterated branched chain amino acids (and their oxo-acid derivatives) were incorporated into branched C₄ and C₅ acid groups of glucose esters by a process of transamination, oxidative decarboxylation and subsequent acylation. C₄ and C₅ branched acids were elongated by two carbon units to produce the branched C₁₀-C₁₂ groups. Norvaline, norleucine, allylglycine, and methionine also were processed into acyl moieties and secreted from the trichomes as glucose esters. Changes in the acyl composition of the glucose esters following sulfonylurea herbicide administration support the participation of acetohydroxyacid synthetase and the other enzymes of branched amino acid biosynthesis in the production of glucose esters.     

A rapid method for isolating glandular trichomes

A physical method is described for the rapid isolation of plant trichomes, with emphasis on stalked glandular types. The technique involved breaking frozen trichomes with powdered dry ice and collection of glandular heads by sieving from larger tissue fragments. This method was applied to several plants that bear similar stalked trichomes: geranium (Pelargonium), potato (Solanum tuberosum), tomato (Lycopersicon esculentum), squash (Cucurbita pepo), and velvetleaf (Abutilon theophrasti). The tissue preparation was of sufficient quality without further purification for biochemical and molecular studies. The preparation maintained the biochemical integrity of the trichomes for active enzymes and usable nucleic acids. A large quantity of tissue can be harvested; for example, 351 milligrams dry weight of glandular trichomes were harvested from geranium pedicels in 12 hours. The utility of the technique was demonstrated by examining the fatty acid composition of tall glandular trichomes of geraniums, Pelargonium ×hortorum L.H. Bailey. These purified cells contained high concentrations of unusual ω5-unsaturated fatty acids, proportionally 23.4% of total fatty acids in the trichomes. When the trichomes were removed, the supporting tissue contained no ω5-fatty acids, thereby unequivocally localizing ω5-fatty acids to the trichomes. Because ω5-fatty acids are unique precursors for the biosynthesis of ω5-anacardic acids, we conclude that anacardic acid synthesis must occur in the glandular trichomes.     

Engineering of tomato type VI glandular trichomes for trans-chrysanthemic acid biosynthesis,the acid moiety of natural pyrethrin insecticides

Glandular trichomes, known as metabolic cell factories, have been proposed as highly suitable for metabolically engineering the production of plant high-value specialized metabolites. Natural pyrethrins, found only in Dalmatian pyrethrum (Tanacetum cinerariifolium), are insecticides with low mammalian toxicity and short environmental persistence. Type I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of the three rethrolone-type alcohols. To test if glandular trichomes can be made to synthesize trans-chrysanthemic acid, we reconstructed its biosynthetic pathway in tomato type VI glandular trichomes, which produce large amounts of terpenoids that share the precursor dimethylallyl diphosphate (DMAPP) with this acid. This was achieved by coexpressing the trans-chrysanthemic acid pathway related genes including TcCDS encoding chrysanthemyl diphosphate synthase and the fusion gene of TcADH2 encoding the alcohol dehydrogenase 2 linked with TcALDH1 encoding the aldehyde dehydrogenase 1 under the control of a newly identified type VI glandular trichome-specific metallocarboxypeptidase inhibitor promoter. Whole tomato leaves harboring type VI glandular trichomes expressing all three aformentioned genes had a concentration of total trans-chrysanthemic acid that was about 1.5-fold higher (by mole number) than the levels of β-phellandrene, the dominant monoterpene present in non-transgenic leaves, while the levels of β-phellandrene and the representative sesquiterpene β-caryophyllene in transgenic leaves were reduced by 96% and 81%, respectively. These results suggest that the tomato type VI glandular trichome is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.     

An ethylene-forming enzyme in Citrus unshiu fruits

An ethylene-forming enzyme from Citrus unshiu fruits was purified some 630-fold. The enzyme catalysed ethylene formation from 1-aminocyclopropane-1-carboxylic acid in the presence of pyridoxal phosphate, β-indoleacetic acid, Mn²⁺ and 2,4-dichlorophenol. It behaved as a protein of MW 40 000 on Sephacryl S-200 gel filtration, and gave one band corresponding to a MW of 25 000 on SDS-PAGE. It had a specific activity of 0.025 μmol/min·mg protein. It exhibited IAA oxidase activity and had no guaiacol peroxidase or NADH oxidase activity. Its K_m for ACC was 2.8 mM, and its pH optimum was 5.7. It was inhibited by potassium cyanide n-propyl gallate and Tiron. d-Mannose, histidine, iodoacetate, PCMB, dimethylfuran and superoxide dismutase showed no inhibition. β-Indoleacrylic acid against IAA competitively inhibited ethylene formation. Other IAA analogues, such as β-indolepropionic acid, β-indolecarboxylic acid and β-indolebutylic acid, slightly stimulated ethylene formation. β-Indoleacrylic acid against 1-aminocyclopropane-1-carboxylic acid non-competitively inhibited ethylene formation. Ascorbate was a potent inhibitor. The inhibitory effects, however, were not always reproduced in vivo. It is difficult to identify this enzyme system as a natural in vivo system from the above observations. Nevertheless, the possible in vivo participation of this in vitro enzyme system is discussed.     

Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6–10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C₆–C₁₀ carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes. Using this approach, the expression of the Pseudomonas putida alkane monooxygenase system, encoded by alkBGT, in combination with all β-oxidation reversal enzymes resulted in the production of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid. Following identification and characterization of potential alcohol and aldehyde dehydrogenases, chnD and chnE from Acinetobacter sp. strain SE19 were expressed in conjunction with alkBGT to demonstrate the synthesis of the C₆–C₁₀ dicarboxylic acids, adipic acid, suberic acid, and sebacic acid. The potential of a β-oxidation cycle with ω-oxidation termination pathways was further demonstrated through the production of greater than 0.8 g/L C₆–C₁₀ ω-hydroxyacids or about 0.5 g/L dicarboxylic acids of the same chain lengths from glycerol (an unrelated carbon source) using minimal media.     

Identification of the acid/base catalyst of a glycoside hydrolase family 3 (GH3) β-glucosidase from Aspergillus niger ASKU28

Background

The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined.

Methods

In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28.

Results

In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced k_cat and k_cat/K_m values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product.

Conclusions and general significance

These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members.     

Arabidopsis ketoacyl‐CoA synthase 16 (KCS16) forms C36/C38 acyl precursors for leaf trichome and pavement surface wax

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a β‐ketoacyl‐CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C₃₅ and C₃₇ alkanes derived from C₃₆ and C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss‐of‐function mutants were depleted of C₃₅ and C₃₇ alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C₃₆ and C₃₈ fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C₃₄ to C₃₈ acyl‐CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra‐long compounds in adjacent pavement cells.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

The complete amino acid sequences of the β1- and β2-subunits of the isolectins LoL1 and LoL11 from seeds of Lathyrus ochrus (L.) DC

The complete amino acid sequences of the β₁- and β₂-subunits of the isolectins (LoL1 and LoL11) from seeds of Lathyrus ochrus were determined by analysis of peptides derived from the proteins by digestion with trypsin, chymotrypsin, pepsin and the S. aureus V₈ protease, as well as fragments produced by cleavage with iodosobenzoic acid. Both β-subunits consisted of singlepolypeptide chains of 181 amino acids, which differed from one another in only 3 positions. The homology of the Lathyrus ochrus isolectins with the other two-chain lectins of the tribe Vicieae, and the single-chain lectins of other tribes of the Leguminosae is discussed.     

Rapid β-oxidation of eicosapentaenoic acid in mouse brain: An in situ study

Analyses of brain phospholipid fatty acid profiles reveal a selective deficiency and enrichment in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively. In order to account for this difference in brain fatty acid levels, we hypothesized that EPA is more rapidly β-oxidized upon its entry into the brain. Wild-type C57BL/6 mice were perfused with either ¹⁴C-EPA or ¹⁴C-DHA via in situ cerebral perfusion for 40 s, followed by a bicarbonate buffer to wash out the residual radiolabeled polyunsaturated fatty acid (PUFA) in the capillaries. ¹⁴C-PUFA-perfused brains were extracted for chemical analyses of neutral lipid and phospholipid fatty acids. Based on the radioactivity in aqueous, total lipid, neutral lipid and phospholipid fractions, volume of distribution (V_D, μl/g) was calculated. The V_D between ¹⁴C-EPA- and ¹⁴C-DHA-perfused samples was not statistically different for total lipid, neutral lipids or total phospholipids. However, the V_D of ¹⁴C-EPA in the aqueous fraction was 2.5 times higher than that of ¹⁴C-DHA (p=0.025), suggesting a more extensive β-oxidation than DHA. Furthermore, radiolabeled palmitoleic acid, a fatty acid that can be synthesized de novo, was detected in brain phospholipids from ¹⁴C-EPA but not from ¹⁴C-DHA-perfused mice suggesting that β-oxidation products of EPA were recycled into endogenous fatty acid biosynthetic pathways. These findings suggest that low levels of EPA in brain phospholipids compared to DHA may be the result of its rapid β-oxidation upon uptake by the brain.     

A recruited protease is involved in catabolism of pyrimidines

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-β-alanine to β-alanine, is catalyzed by two β-alanine synthase (βASase, EC 3.5.1.6) subfamilies. We show that the “prototype” eukaryote βASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-βA compared with a representative of fungal βASases, the yeast Saccharomyces kluyveri βASase, which has a high K_m value (71 mM). S. kluyveri βASase is specifically inhibited by dipeptides and tripeptides, and the apparent K_i value of glycyl-glycine is in the same range as the substrate K_m. We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal βASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.     

Vitamin K1 dependent carboxylase: β-Carboxylation of t-butyloxycarbonylaspartic acid α-benzyl ester

Rat liver microsomes solubilized by Triton X-100 catalyze the vitamin K₁ dependent incorporation of carbon-14 from [¹⁴C]NaHCO₃ into t-butyloxycarbonylaspartic acid α-benzyl ester. High voltage electrophoresis of the alkaline hydrolysate of the products of this reaction demonstrates the presence of a labelled species (A) whose electrophoretic mobility is identical to that of β-carboxyaspartic acid. High voltage electrophoresis of the acid-treated products reveals the disappearance of A and the appearance of a labelled species whose electrophoretic mobility is identical to that of aspartic acid. These experiments provide unequivocal evidence for the vitamin K₁ dependent β-carboxylation of an aspartic acid side chain, and they constitute the first report of such an enzymatic activity in microsomes.     

Trypanosoma rangeli: An alkaline ecto-phosphatase activity is involved with survival and growth of the parasite

The aim of this work was to investigate whether an alkaline ecto-phosphatase activity is present in the surface of Trypanosoma rangeli. Intact short epimastigote forms were assayed for ecto-phosphatase activity to study kinetics and modulators using β-glycerophosphate (β-GP) and p-nitrophenyl phosphate (pNPP) as substrates. Its role in parasite development and differentiation was also studied. Competition assays using different proportions of β-GP and pNPP evidenced the existence of independent and non-interacting alkaline and acid phosphatases. Hydrolysis of β-GP increased progressively with pH, whereas the opposite was evident using pNPP. The alkaline enzyme was inhibited by levamisole in a non-competitive fashion. The Ca²⁺ present in the reaction medium was enough for full activity. Pretreatment with PI-PLC decreased the alkaline but not the acid phosphatase evidence that the former is catalyzed by a GPI-anchored enzyme, with potential intracellular signaling ability. β-GP supported the growth and differentiation of T. rangeli to the same extent as high orthophosphate (P_i). Levamisole at the IC₅₀ spared significantly parasite growth when β-GP was the sole source of P_i and stopped it in the absence of β-GP, indicating that the alkaline enzyme can utilize phosphate monoesters present in serum. These results demonstrate the existence of an alkaline ecto-phosphatase in T. rangeli with selective requirements and sensitivity to inhibitors that participates in key metabolic processes in the parasite life cycle.     

Molecular heterogeneity of amyloid β2-microglobulin and modification with advanced glycation end products

By using liquid chromatography–electrospray ionization mass spectrometry, Western blotting and N-terminal amino acid sequence analysis, we characterized the molecular heterogeneity and advanced glycation end product (AGE) modification of β₂-microglobulin (β₂m) extracted from the amyloid tissue of a hemodialysis patient. Amyloid β₂m was composed of full-length β₂m, truncated β₂m and dimer β₂m. Truncated β₂m and dimer β₂m were modified with AGEs such as imidazolone and N^ϵ-(carboxymethyl)lysine, and showed fluorescence characteristic of AGE. Truncated β₂m species were formed by cleavage between amino acid residues of Pro⁶/Ile⁷, Gln⁸/Val⁹ and Val⁹/Tyr¹⁰. Heterogeneous dimer β₂m species showed the molecular masses of 22 591 and 22 675, which resulted from cross-linking between truncated β₂m.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Carbon limitation enhances CO2 concentrating mechanism but reduces trichome size in Arthrospira platensis (cyanobacterium)

Arthrospira species grow well under highly enriched inorganic carbon concentrations, but little is known on the effects of inorganic carbon (Ci) limitation on its physiological performance. When Arthrospira platensis D-0083 was grown in a modified medium without NaHCO₃ under ambient air of 380 ppm CO₂, its trichomes became disassembled while the growth and photosynthetic rates were severely reduced. Phycocyanin and allophycocyanin contents decreased but the carotenoid content increased under the Ci limitation. Compared with the cells grown in Zarrouk medium, the trichomes grown under the Ci limitation increased their photosynthetic apparent affinity for Ci by about 14 times but photochemical quenching capacity was reduced. It appeared that A. platensis increased its CO₂ concentrating mechanism by inducing HCO₃ ^? transporters and reducing the trichome size which increased filamentous surface to volume ratio.     

Occurrence of Diatom – Diazotrophic association in the coastal surface waters of south Andaman,India

The heterocystous cyanobacterium Richelia intracellularis Schmidt 1901 is well known for its capability to fix atmospheric nitrogen (N₂) in oligotrophic waters. Symbiotic associations of Richelia intracellularis with the diatoms Rhizosolenia hebetata, Rhizosolenia clevei, Rhizosolenia cylindrus and Hemiaulus membranaceus is reported for the first time from the coastal waters of South Andaman, India. In these the symbiotic associations, variations were observed in the number of vegetative cells, trichomes, and the shape of the heterocysts. The highest number of trichomes was observed in Rhizosolenia clevei (14) per host. The trichomes consisted of 9–10 vegetative cells and a spherical heterocyst that was orientiated toward one end of the host diatom cell. In Rhizosolenia hebetata, three trichomes of Richelia intracellularis were present, one trichome at one end with 7–8 vegetative cells and two trichomes at the other end. In Rhizosolenia cylindrus, two trichomes of Richelia were observed, with a spherical heterocyst and 5–6 vegetative cells. An association between Hemiaulus membranaceus and Richelia intracellularis was also observed but with less frequency. Trichomes were observed in the centre of the diatom. By using the cell-specific rates, the amount of new N provided by the Diatom-Diazotrophic Associations was estimated. This is the first record of a symbiotic association of Richelia in diatoms from Andaman coastal waters.     

Long chain esters of Virola species

The esters of n-fatty acids and ω-hydroxy n-fatty acids of β-sitosterol, D-glucose and ferulic acid (trans and cis) as well as β-sitosterol, fatty acids and β-sitosteryl-β-D-glucoside were isolated from three Virola species and identified by optical data and chemical reactions. A novel series of acidic esters derived from C₂₂C₂₉ ω-hydroxy fatty acids and cis- and trans-ferulic acid is reported for the first time. These compounds also occurred as the corresponding diester 1-monoglycerides whereas the ω-hydroxy acids themselves were also present as the corresponding glucosyl esters.