New 4-O-substituted xylosyl units in the xyloglucan from leaves of Hymenaea courbaril

A homogeneous fucogalactoxyloglucan, isolated from the leaves of Hymenaea courbaril, was analysed by methylation-GC–MS. These procedures involved derived partially O-methylated alditol acetates and acetylated aldononitriles, which demonstrated the presence of both 2-O- and 4-O-substituted Xylp units in the side-chains. The presence of the unusual, latter structure was confirmed by 2D NMR spectroscopy with a correlated HMQC C-4/H-4 signal at δ 77.8/3.73. A similar 4-O-substituted xylosyl structure was present in a decasaccharide Glc₄Xyl₃Gal₂Fuc obtained via endo-glucanase treatment of the polysaccharide, which gave rise to a molecular ion with m/z 1555 (ESI-MS, Na⁺ form).     

Xyloglucan Octasaccharide XXLGol Derived from the Seeds of Hymenaea courbaril Acts as a Signaling Molecule

Treatment of the xyloglucan isolated from the seeds of Hymenaea courbaril with Humicola insolens endo-1,4-β-d-glucanase I produced xyloglucan oligosaccharides, which were then isolated and characterized. The two most abundant compounds were the heptasaccharide (XXXG) and the octasaccharide (XXLG), which were examined by reference to the biological activity of other structurally related xyloglucan compounds. The reduced oligomer (XXLGol) was shown to promote growth of wheat (Triticum aestivum) coleoptiles independently of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D). In the presence of 2,4-D, XXLGol at nanomolar concentrations increased the auxin-induced response. It was found that XXLGol is a signaling molecule, since it has the ability to induce, at nanomolar concentrations, a rapid increase in an α-l-fucosidase response in suspended cells or protoplasts of Rubus fruticosus L. and to modulate 2,4-D or gibberellic acid-induced α-l-fucosidase.     

Ipomopsin and hymenain,two biscoumarins from seeds of Hymenaea courbaril

A new biscoumarin (7-hydroxy-6,6′-dimethoxy-3,7′-dicoumarinyl ether), named hymenain (1) and the known biscoumarin ipomopsin (2) were isolated from the germinated seeds of Hymenaea courbaril var. stilbocarpa. Their structures were elucidated by spectroscopic methods. Scopoletin was also detected in the extracts by TLC and GC–MS analyses. Hymenain and ipomopsin showed direct scavenging effect on a stable free radical, 2,2-diphenyl-1-picryl-hydrazyl (DPPH) with the EC₅₀ values of 100 and 300 μM, respectively.     

Clerodane-type diterpenes from the seed pods of Hymenaea courbaril var. stilbocarpa.

Three known and two new diterpenes were isolated from the ethyl acetate extract of Hymenaea courbaril var. stilbocarpa seed pods. One of the compounds was elucidated as (5R*,8S*,9S*,10R*)-cleroda-3,13E-dien-15-oic acid and the other was elucidated, after treatment with diazomethane, as methyl (5S*,8S*,9S*,10R*)-cleroda-3,13E-dien-15-oate. The known diterpenes were identified as (-)-ozic acid, (-)-isoozic acid and (-)-kovalenic acid which were characterized as their methyl ester derivatives.     

Microsatellite loci isolated from the tropical tree Hymenaea courbaril L. (Fabaceae)

We developed 11 microsatellite markers for Hymenaea courbaril for the purpose of studying spatial genetic structure and gene flow. The microsatellite loci were screened in 44 trees from two populations. All loci were polymorphic, exhibiting between two and 16 alleles, and levels of expected heterozygosity from 0.174 to 0.909. Departures from Hardy-Weinberg equilibrium were detected for all loci in one population. The estimated null allele frequency is low or moderate. No locus combinations exhibited linkage disequilibrium.     

Phylogeographical structure of the neotropical forest tree Hymenaea courbaril (Leguminosae: Caesalpinioideae) and its relationship with the Vicariant Hymenaea stigonocarpa from Cerrado

The phylogeography of Hymenaea courbaril var. stilbocarpa from Atlantic Forest and riverine forests of the Cerrado biome in central and southeastern Brazil was investigated. The data were compared with those of its congeneric Hymenaea stigonocarpa, a typical tree from savanna. In the Cerrado, H. courbaril var. stilbocarpa is found in sites contiguous with those of H. stigonocarpa, and they share common life-history attributes. The psbC/trnS3 region of the chloroplast DNA was sequenced in 149 individuals of H. courbaril var. stilbocarpa. High genetic variation was found in this species, with the identification of 18 haplotypes, similarly to what was found in H. stigonocarpa with 23 haplotypes in the same geographic region. Populations of H. courbaril var. stilbocarpa could be structured in 3 phylogeographic groups. Spatial analysis of molecular variation indicated that 46.4% of the genetic variation was due to differences among these groups. Three haplotypes were shared by H. courbaril var. stilbocarpa and H. stigonocarpa, and only 10.5% of the total genetic variation could be attributed to between-species difference. We surmise that during the glacial times, H. courbaril var. stilbocarpa populations must have gone extinct in most parts of the southern of its present-day occurrence area. After climate amelioration, these areas were probably recolonized from northern and eastern. The relatively similar phylogeographic structure of vicariant Hymenaea species suggests that they were subjected to the same impacts during the Quaternary climatic fluctuations. The sharing of haplotypes and the genetic similarity between the 2 Hymenaea species suggest the existence of ancestral polymorphism and/or hybridization.     

The control of storage xyloglucan mobilization in cotyledons of Hymenaea courbaril

Hymenaea courbaril is a leguminous tree species from the neotropical rain forests. Its cotyledons are largely enriched with a storage cell wall polysaccharide (xyloglucan). Studies of cell wall storage polymers have been focused mostly on the mechanisms of their disassembly, whereas the control of their mobilization and the relationship between their metabolism and seedling development is not well understood. Here, we show that xyloglucan mobilization is strictly controlled by the development of first leaves of the seedling, with the start of its degradation occurring after the beginning of eophyll (first leaves) expansion. During the period of storage mobilization, an increase in the levels of xyloglucan hydrolases, starch, and free sugars were observed in the cotyledons. Xyloglucan mobilization was inhibited by shoot excision, darkness, and by treatment with the auxin-transport inhibitor N-1-naphthylphthalamic acid. Analyses of endogenous indole-3-acetic acid in the cotyledons revealed that its increase in concentration is followed by the rise in xyloglucan hydrolase activities, indicating that auxin is directly related to xyloglucan mobilization. Cotyledons detached during xyloglucan mobilization and treated with 2,4-dichlorophenoxyacetic acid showed a similar mobilization rate as in attached cotyledons. This hormonal control is probably essential for the ecophysiological performance of this species in their natural environment since it is the main factor responsible for promoting synchronism between shoot growth and reserve degradation. This is likely to increase the efficiency of carbon reserves utilization by the growing seedling in the understorey light conditions of the rain forest.     

Isolation and characterization of microsatellite loci for Hymenaea courbaril and transferability to Hymenaea stigonocarpa, two tropical timber species

Hymenaea courbaril is a tropical timber species, intensely exploited and found in the Amazon, Atlantic Forest and Brazilian Cerrado biome. Nine highly polymorphic microsatellite loci were developed from a genomic library enriched for AG/TC repeats. In a total of 41 individuals, from two natural populations, seven to 13 alleles per locus were detected and expected heterozygosity ranged from 0.75 to 0.90. Seven loci were effectively transferred to Hymenaea stigonocarpa. High levels of polymorphism make the present primers useful for population genetic studies and are a powerful tool to investigate mating system, gene flow and spatial genetic structure.     

Mendelian inheritance, linkage and genotypic disequilibrium in microsatellite loci isolated from Hymenaea courbaril (Leguminosae)

The Neotropical tree Hymenaea courbaril, locally known as Jatobá, is a valuable source of lumber and also produces comestible and medicinal fruit. We characterized Mendelian inheritance, linkage and genotypic disequilibrium at nine microsatellite loci isolated from H. courbaril, in order to determine if they would provide accurate estimates of population genetic parameters of this important Amazon species. The study was made on 250 open-pollinated offspring originated from 14 seed trees. Only one of nine loci presented significant deviation from the expected Mendelian segregation (1:1). Genotypic disequilibrium between pairwise loci was investigated based on samples from 55 adult and 56 juvenile trees. No genetic linkage between any paired loci was observed. After Bonferroni's corrections for multiple tests, we found no evidence of genotypic disequilibrium between pairs of loci. We conclude that this set of loci can be used for genetic diversity/ structure, mating system, gene flow, and parentage analyses in H. courbaril populations.     

The role of the storage carbon of cotyledons in the establishment of seedlings of Hymenaea courbaril under different light conditions

BACKGROUND AND AIMS: Hymenaea courbaril (Leguminosae-Caesalpinioideae) is a tree species with wide distribution through all of the Neotropics. It has large seeds (approx. 5 g) with non-photosynthetic storage cotyledons rich (40 %) in a cell wall polysaccharide (xyloglucan) as a carbon reserve. Because it is found in the understorey of tropical forests, it has been considered as a shade-tolerant, late-secondary species. However, the physiological mechanisms involved in seedling establishment, especially regarding the interplay between storage and light intensity, are not understood. In this work, the ecophysiological role of this carbon cotyledon reserve (xyloglucan) is characterized, emphasizing its effects on seedling growth and development during the transition from heterotrophy to autotrophy under different light conditions. METHODS: Seedlings of H. courbaril were grown in environments with different light intensities, and with or without cotyledons detached before xyloglucan mobilization. Development, growth, photosynthesis and carbon partitioning (dry mass and [14C]sucrose) were analysed in each treatment. KEY RESULTS: The detachment of cotyledons was not important for seedling survival, but resulted in a strong restriction (50 % less) of shoot growth, which was the main sink for the cotyledon carbon reserves. Carbon restriction promoted an early maturation of the photosynthetic apparatus without changes in the net CO2 fixation per unit area. The reduced surface area of the first leaves in seedlings without cotyledons was evidence of limited growth and development of seedlings in low light conditions (22 micromol m(-2) s(-1) photon flux). CONCLUSIONS: There is an increase in the importance of storage xyloglucan in cotyledons for H. courbaril seedling development as light intensity decreases, confirming that this polymer plays a key role in the adaptation of this species to establish successfully in the shadowed understorey of the forest.     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Localization of Xyloglucan in the Macromolecular Complex Composed of Xyloglucan and Cellulose in Pea Stems

A macromolecular complex composed of xyloglucan and cellulosewas isolated from elongating regions of stems of etiolated pea(Pisum sativum L. var Alaska) seedlings and binding of a xyloglucan-specificantibody was examined after treatment of the complex with endo-1,4-ß-glucanaseor 24% KOH. The antibody bound to the complex but the extentof binding was reduced after treatment of the complex with endo-1,4-ß-glucanaseand was hardly detectable after treatment with 24% KOH. Themolecular weight of the xyloglucan that remained (5%) in theß-glucanase-treated complexes was less than 9,200.Pea xyloglucan was allowed to bind to enzymeand alkali-treatedcomplexes to generaly reconstituted complexes. The amount ofthe antibody that bound to each type of reconstituted complexwas similar but was much lower than that bound to the nativecomplex. Immunogold labeling indicated that most of the antigenwas widely distributed between microfibrils in the native complex,whereas the antigen appeared to be confined to the microfibrilsin the reconstituted complexes. These findings suggest thata part of each xyloglucan molecule is strongly associated withcellulose microfibrils while the rest is free of the microfibrilsin the native complex. ¹This work was supported in part by a grant from the YamadaScience Foundation.     

Characterization of Kiwifruit Xyloglucan

Structural characteristics of xyloglucan are constant in the pericarp cell walls of kiwifruit (Actinidia deliciosa) throughout fruit enlargement and maturation. Most of the xyloglucan (XG) persists in the cell walls of ripe kiwifruit. XG from the pericarp tissues of 36-h ethylene-treated kiwifruit was extracted as hemicellulose Ⅱ (HC-Ⅱ) with 4.28 M KOH containing 0.02% NaBH4, and purified using iodine precipitation and subsequent anion-exchange chromatography. This purifying protocol increased XG purity from 50 mol% in HC-Ⅱ fraction to 62 mol% in the purified XG powder. The molar ratio of glucose: xylose: galactose: fucose in the purified XG was 10: 6.9: 2.1: 0.3. Gel permeation chromatography indicated that purified XG had an average molecular-mass of 161 KDa, a value that exceeds the 95 KDa M_r determined for total polymeric sugars. Sugar linkage analysis confirmed the lack of fucose in the kiwifruit XG, but a small amount of arabinoxylan and low M_r glucomannan remained associated with this fraction.     

Strong correlation between isoprene emission and gross photosynthetic capacity during leaf phenology of the tropical tree species Hymenaea courbaril with fundamental changes in volatile organic compounds emission composition during early leaf development

Changes of the volatile organic compounds (VOC) emission capacity and composition of different developmental stages of the tropical tree species Hymenaea courbaril were investigated under field conditions at a remote Amazonian rainforest site. The basal emission capacity of isoprene changed considerably over the course of leaf development, from young to mature and to senescent leaves, ultimately spanning a wide range of observed isoprene basal emission capacities from 0.7 to 111.5 µg C g^?1 h^?1 during the course of the year. By adjusting the standard emission factors for individual days, the diel courses of instantaneous isoprene emission rates could nevertheless adequately be modelled by a current isoprene algorithm. The results demonstrate the inadequacy of using one single standard emission factor to represent the VOC emission capacity of tropical vegetation for an entire seasonal cycle. A strong linear correlation between the isoprene emission capacity and the gross photosynthetic capacity (GP_max) covering all developmental stages and seasons was observed. The present results provide evidence that leaf photosynthetic properties may confer a valuable basis to model the seasonal variation of isoprenoid emission capacity; especially in tropical regions where the environmental conditions vary less than in temperate regions. In addition to induction and variability of isoprene emission during early leaf development, considerable amounts of monoterpenes were emitted in a light‐dependent manner exclusively in the period between bud break and leaf maturity. The fundamental change in emission composition during this stage as a consequence of resource availability (supply side control) or as a plant's response to the higher defence demand of young emerging leaves (demand‐side control) is discussed. The finding of a temporary emergence of monoterpene emission may be of general interest in understanding both the ecological functions of isoprenoid production and the regulatory processes involved.     

The Structure and Functions of Xyloglucan

Xyloglucan is a polysaccharide found in the primary cell wallsof all higher plants examined. Its cellulose-like backbone,which is about 0.15 to 1.5 µm long, consists of 300 to3 000 ß-(14)-linked D-glucopyranose residues. About60–75% (or, in grasses, about 30–40%) of the glucoseresidues have side-chains attached to position 6. The majorside-chains are: D-xylopyranosyl--1 -, D-galactopyranosyl-ß-(12)-D-xylopyranosyl--I , L-arabinofuranosyl-(1 -2)-D-xylopyranosyl--1-, and (except in grasses) L.-fucopyranosyl--(1 -2)-D-galactopyranosyl-ß-(1-2)-D-xylopyranosyl--1-. There is some regularity in the distribution of these side-chainsalong the backbone. Xyloglucan plays two very different r?les in the control ofcell growth: (a) as a major building material of the wall [concentrationof xyloglucan in the wall in vivo 10% (w/v)] it probably directlydictates wall extensibility and, therefore, the rate of cellexpansion and (b) it can be broken down to a fucose-containingoligosaccharide which [at a concentration of 0.0000001% (w/v)]exerts a hormone-like anti-auxin effect on growth. In addition,xyloglucan lacking fucose is used by certain dicotyledonousseeds as a food reserve which is mobilized after germination.Xyloglucan is, therefore, the subject of considerable currentinterest in several apparently disparate areas of botany. Key words: Xyloglucan, ‘oligosaccharin’, hemicellulose, auxin, anti-auxin, growth, cell walls, reserve carbohydrate     

Xyloglucan galactosyl- and fucosyltransferase activities from pea epicotyl microsomes.

Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific beta-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs.     

Degradation of Xyloglucan by Wall-Bound Enzymes from Soybean Tissue II: Degradation of the Fragment Heptasaccharide from Xyloglucan and the Characteristic Action Pattern of the {alpha}-D-Xylosidase in the Enzyme System

Hydrolysis of the fragment heptasaccharide (glucose : xylose= 4 : 3) from xyloglucan with an enzyme preparation from soybeancell wall produced a penta- and a trisaccharide. The resultsof fragmentation analysis of these oligosaccharides with Aspergillusoryzae ß-D-glucosidase indicate the following structuresfor the penta- and trisaccharide. The detection of these intermediate products suggested thatdegradation of the heptasaccharide took place by sequentialsplitting of the -D-xylosidic and ß-D-glucosidic linkages.A characteristic action pattern of the a-D-xylosidase in theenzyme preparation was found. ¹Present address: Department of Biology, McGill University,Montreal, Canada. ²Present address: Department of Botany, Iowa State University,Ames, Iowa 50010, U.S.A. (Received August 20, 1982; Accepted December 7, 1982)     

Measurement of Oligosaccharides Derived from Tamarind Xyloglucan by Competitive ELISA Assay

Xyloglucan oligosaccharides, which were derived from tamarind xyloglucan by cellulase digestion, were measured by a competitive ELISA method using antibodies raised against a complex of BSA and xyloglucan nonasaccharide. By this method, tamarind xyloglucan oligosaccharides could be measured in the range of 0.1 to 40 nmol/well.