Protoplast isolation and culture from carob (Ceratonia siliqua) hypocotyls: ability of regenerated protoplasts to produce mannose-containing polysaccharides

The aim of this study was to isolate protoplasts from carob (Ceratonia siliqua L.) embryonic tissues with the ability to regenerate cell walls, divide and synthesize galactomannan, a valuable polysaccharide for industry. Protoplasts isolated from carob hypocotyl hooks regenerated cell walls within 24 h. The first divisions of the regenerated cells were observed after 2 days of culture. The highest percentage that successfully divided was achieved when the seedlings were grown under diffuse light, the hypocotyl hooks were plasmolysed for 1 h before incubation in the protoplast isolation solution and the protoplasts were cultured under diffuse light. After 9 days of culture, cell clusters, consisting of eight cells, had been produced, which underwent further mitotic divisions and which were expected to lead to callus formation. Polysaccharide and oligosaccharide synthesis during protoplast regeneration was studied by radiolabelling with exogenous d ‐[U‐¹⁴C]glucose, d ‐[U‐¹⁴C]mannose or d ‐[2‐³H]mannose, which gave rise to uniform, moderately specific and highly specific labelling, respectively. As revealed by the radioactivity distribution in cell wall monosaccharides, the regenerants deposited new wall polymers that differed markedly from those being synthesized by the hypocotyls from which the protoplasts had been isolated. The regenerants deposited large amounts of callose and smaller amounts of galactose‐, arabinose‐ and mannose‐containing polymers. The latter included glucuronomannan, as demonstrated by a new method involving partial acid hydrolysis followed by β‐glucuronidase (EC 3.2.1.31) digestion. The regenerating protoplasts also released soluble extracellular carbohydrates: polysaccharides which appeared to be mainly acidic arabinogalactans, and oligosaccharides which were mainly neutral and contained glucose, galactose and mannose. We conclude that regenerating carob protoplasts are a useful system for studying carbohydrate secretion, including mannose‐rich poly‐ and oligosaccharides.     

Galactomannan formation and guanosine 5′-diphosphate-mannose: galactomannan mannosyltransferase in developing seeds of fenugreek (Trigonella foenum-graecum L., leguminosae)

The time-course of galactomannan and stachyose (digalactosyl-sucrose) deposition in the fenugreek seed endosperm has been determined, and correlated with standard parameters of seed development. During, and only during, the period of galactomannan deposition, endosperm homogenates are capable of catalysing the transfer of labelled d-mannosyl residues from guanosine 5-diphosphate d-[U-¹⁴C]mannose to a soluble polysaccharide product indistinguishable from galactomannan. The mannosyltransferase activity peaks twice, once at the beginning of galactomannan deposition, and again in the middle of the most rapid phase of galactomannan deposition. The enzyme in the later peak sediments with grossly particulate material (1,000 g pellet), whereas the earlier peak contains a considerable proportion of a particulate enzyme sedimenting at 100,000 g. These observations are discussed in the light of existing information on the ultrastructural aspects of galactomannan deposition. The mannosyltransferase is clearly involved in galactomannan formation in vivo, but the status of an accompanying galactosyltransferase is less clear.Abbreviations GDP guanosine 5-diphosphate - UDP uridine 5-diphosphate     

Production and secretion of {alpha}-galactosidase and endo-{beta}-mannanase by carob (Ceratonia siliqua L.) endosperm protoplasts

Viable protoplasts were isolated for the first time from maturecarob (Ceratonia siliqua L.) endosperm tissue. After 5 d ofincubation 75% of the protoplasts were viable. During incubationthey underwent vacuolation and produced the carob endospermhydrolases, agalactosidase and endo-ß-mannanase, whichwere secreted in the incubation medium. The secretion of bothenzymes were under Ca²⁺ control. Many characteristics of -galactosidaseand endo-ß-mannanase production by protoplasts werethe same as those of whole endosperms: their production didnot require any hormonal signal and was inhibited in the presenceof ABA or the leachate from the carob endosperm/seed coat. Moderatewater stress (—2.0 MPa) neither affected the activityof these hydrolases nor their secretion by endosperm protoplast.However, when the osmoticum of protoplast incubation mediumwas higher, the production and secretion of both hydrolaseswere reduced. Comparison of the hydrolases activities in theincubation media of leached carob endosperms, which were incubatedunder normal and water stress (—1.5 MPa) conditions, withthe activities of the protoplast-secreted hydrolases indicatedthat (i) carob endosperm cell wall acts as a barrier for thesecreted enzymes and (ii) that water stress reduces the cellwall porosity of the carob endosperm cells, and thus the releaseof the secreted -galactosidase and endo-ß-mannanaseis inhibited. The isolation of carob endosperm protoplasts offersa potent experimental system for the study of aspects of endospermcell physiology, such as enzyme secretion Key words: Abscisic acid, carob endosperm, Ceratonia siliqua L, endo-ß-mannanase, -galactosidase, leachate, protoplasts, water stress     

Control of mannose/galactose ratio during galactomannan formation in developing legume seeds

Galactomannan deposition was investigated in developing endosperms of three leguminous species representative of taxonomic groups which have galactomannans with high, medium and low galactose content. These were fenugreek (Trigonella foenum-graecum L.; mannose/galactose (Man/Gal) = 1.1), guar (Cyamopsis tetragonoloba (L.) Taub.; Man/Gal = 1.6) and Senna occidentalis (L.) Link. (Man/Gal = 3.3), respectively. Endosperms were analysed at different stages of seed development for galactomannan content and the levels, in cell-free extracts, of a mannosyltransferase and a galactosyltransferase which have been shown to catalyse galactomannan biosynthesis in vitro (M. Edwards et al., 1989, Planta 178, 41–51). There was a close correlation in each case between the levels of the biosynthetic mannosyl- and galactosyltransferases and the deposition of galactomannan. The relative in vitro activities of the mannosyl- and galactosyltransferases in fenugreek and guar were similar, and almost constant throughout the period of galactomannan deposition. In Senna the ratio mannosyltransferase/galactosyltransferase was always higher than in the other two species, and it increased substantially throughout the period of galactomannan deposition. In fenugreek and guar the galactomannans present in the endosperms of seeds at different stages of development had the Man/Gal ratios characteristic of the mature seeds. By contrast the galactomannan present in Senna endosperms at the earliest stages of deposition had a Man/Gal ratio of about 2.3. During late deposition this ratio increased rapidly, stabilising at about 3.3, the ratio characteristic of the mature seed. The levels of -galactosidase in the developing endosperms of fenugreek and guar were low and remained fairly constant throughout the deposition of the galactomannan. In Senna, -galactosidase activity in the endosperm was low during early galactomannan deposition, but increased subsequently, peaking during late galactomannan deposition. The developmental patterns of the -galactosidase activity and of the increase in Man/Gal ratio of the Senna galactomannan were closely similar, indicating a cause-and-effect relationship. The endosperm -galactosidase activity in Senna was capable, in vitro, of removing galactose from guar galactomannan without prior depolymerisation of the molecule. In fenugreek and in guar the genetic control of the Man/Gal ratio in galactomannan is not the result of a post-depositional modification, and must reside in the biosynthetic process. In Senna, the Man/Gal ratio of the primary biosynthetic galactomannan product is controlled by the biosynthetic process. Yet the final Man/Gal ratio of the galactomannan in the mature seed is, to an appreciable extent, the result of galactose removal from the primary biosynthetic product by an -galactosidase activity which is present in the endosperm during late galactomannan deposition.Abbreviations al galactose - Man mannose This work was carried out with the aid of a Cooperative Research Grant (No. CRG 1) awarded by the Agricultural and Food Research Council, UK.     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

Galactomannan utilization in germinating legume seeds

Galactose and mannose, released on hydrolysis of galactomannan in the endosperm of germinating seeds of carob, guar, honey locust and lucerne were absorbed by the cotyledons and further metabolized. In guar, the distribution of ¹⁴C from [U-¹⁴C]-d-glucose, d-mannose and D-galactose into various cotyledon fractions did not provide evidence for preferential channelling of d-galactose into cell wall fractions and d-mannose into glycolysis. Phosphomannoisomerase, which has previously been reported in animals and microorganisms was detected in a number of legume seeds. In honey locust it was located in the cotyledons and its level declined after galactomannan was depleted. This enzyme from lucerne was purified until free of phosphoglucoisomerase and some of its properties are described.     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography     

Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants

Yields of 10⁶–10⁸ peach mesophyll cells and protoplasts · gfw^-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4–10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22–30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml^-1. Purification of protoplasts on a sucrose gradient yielded about 10⁷ protoplasts · gfw^-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.Abbreviations BA 6-benzyladenine - IBA indolebutyric acid - FDA fluorescein diacetate - MES 2-M-morpholinoethane sulphonic acid - MS Murashige and Skoog - NAA -naphthaleneacetic acid - PVP polyvinylpyrrolidone     

Ultrastructural and biochemical aspects of cell wall reconstitution in recalcitrant (grapevine) and regenerating (tobacco) leaf protoplasts

Summary To identify possible reasons that may contribute to recalcitrance in plant protoplasts, the time course of new cell wall deposition was studied by scanning electron microscopy in protoplasts of a recalcitrant species, the grapevine. Results showed that microfibrils were developed after 2 days of culture, that complete cell wall formation occurred on Day 6 to 7 of protoplast culture, and its ultrastructural appearance was identical to that of grapevine leaf-derived callus cells. In addition, a comparative study was undertaken on [U-¹⁴C]glucose uptake and incorporation in ethanol-soluble, cellulosic, and noncellulosic polysaccharide fractions in protoplasts of grapevine and of a readily regenerating species, tobacco, during culture. There was a significantly higher [U-¹⁴C]glucose uptake by tobacco than by grapevine protoplasts. The label distribution in the ethanol-soluble, cellulosic, and noncellulosic fractions of newly synthesized cell walls differed quantitatively between the two species. In particular, the labeled glucose incorporated in the noncellulosic cell wall fraction was threefold greater in tobacco than in grapevine protoplasts. Differences were also revealed in the monosaccharide composition of this fraction between the two species. Addition of dimethyl sulfoxide to the culture medium resulted in a dramatic increase in [U-¹⁴C]glucose uptake by grapevine protoplasts, whereas it exhibited a limited effect in tobacco protoplasts. It showed no effect on the ultrastructural characteristics of new cell wall nor on the incorporation rate of labeled glucose in the cellulosic and noncellulosic cell wall fractions.     

Abscisic-acid contents and concentrations in protoplasts from guard cells and mesophyll cells ofVicia faba L.

The abscisic-acid (ABA) contents of isolated guard-cell protoplasts and mesophyll-cell protoplasts fromVicia faba were determined by high-pressure liquid chromatography followed by gas chromatography. The amounts of ABA found immediately after preparation of the protoplasts varied from 90 to 570 amol per guard-cell protoplast, and from 75 to 100 amol per mesophyll-cell protoplast. These contents correspond to concentrations between 36 and 230 mol per liter in guard-cell protoplasts and between 2.7 and 3.3 mol per liter in mesophyll-cell protoplasts. During exposure of protoplasts to betaine concentrations of 0.3, 0.5, and 0.8 mol·l^-1 at 0° and 20°C for 30 min, ABA contents as well as the fractions of ABA that leaked into the medium remained constant for both protoplast types. There was no evidence for net production of ABA in isolated protoplasts subjected to osmotic stress.Abbreviation ABA abscisic acid     

Water stress and galactomannan breakdown in germinated fenugreek seeds. Stress affects the production and the activities in vivo of galactomannan-hydrolysing enzymes

Imposition of water stress on germinated fenugreek (Trigonella foenum-graecum L.) seeds and isolated fenugreek endosperms after the beginning of galactomannan mobilisation caused a reduction in the rate of breakdown of the polysaccharide relative to unstressed controls. The activities, measured in vitro, of the three hydrolytic enzymes involved in the breakdown process (-d-galactosidase, EC 3.2.1.22;endo--d-mannanase, EC 3.2.1.78;exo--d-mannanase, EC 3.2.1.25) were not decreased. Although there was some accumulation of galactomannan-hydrolysis products in endosperms under stress, there was no clear correlation between sugar levels and the inhibition of galactomannan breakdown. When water stress was applied to fenugreek seeds after germination but before the beginning of galactomannan hydrolysis, both galactomannan breakdown and the development of the hydrolytic enzyme activities were inhibited. Washing of newly germinated seeds for 2 h in water prior to the imposition of stress gave partial relief of the inhibition of galactomannan mobilisation, partial recovery ofendo--d-mannanase levels, and full recovery of -d-galactosidase levels. It is argued: 1) that water stress after germination but before the beginning of galactomannan hydrolysis inhibits the production of hydrolytic enzymes in the endosperm, probably via decreased removal at lowered water content of diffusible inhibitory substances; and 2) that water stress after the beginning of galactomannan hydrolysis decreases the rate of galactomannan breakdown in vivo principally via decreased diffusion at lowered water content of enzymes from the aleurone layer through the storage tissue of the endosperm.Abbreviation PEG polyethyleneglycol     

Factors affecting isolation of protoplasts from leaves of grape (Vitis vinifera)

A method of isolating grape mesophyll protoplasts was developed to facilitate the eventual use of genetic engineering techniques in this species. The effects of several factors influencing protoplast isolation could be evaluated quickly by using leaf disks 1 cm in diameter and known volumes of maceration and wash media. The best yields of mesophyll protoplasts were obtained using medium sized leaves of grapevines kept in the dark for 24 hours prior to maceration in 1% Cellulysin, 0.5% Macerase, 0.7 M mannitol, 5 ppm 2, 4 D, 0.1 ppm BAP, 1/10 strength Murashige and Skoog medium, and incubated at 22°C in cool-white fluorescent light (70–100 E m^-2 s^-1) for 24 hours. Over 30×10⁶ protoplasts per cm² of leaf were produced using these conditions. This method of screening factors affecting protoplast isolation could be applicable to other species.     

Isolation and culture of anucleate protoplasts from cotton fiber; assessment of viability and analysis of regenerated wall polymers

Procedures were developed for the isolation and culture of an anucleate protoplast system from cotton fibers actively undergoing secondary wall synthesis. Because the fibers at this stage are elongated single cells (30 m × 1–2 cm), most of the cellular vesicles released in the process of isolation are anucleate. After purification, the protoplast population was nuclei-free. When transferred to culture medium, the anucleate protoplasts (cytoplasts) synthesized starch, hydrolyzed fluorescene diacetate for up to 9 days and formed cell wall material for at least 7 days. The composition of the regenerated cell walls was dependent upon the substrate supplied in the medium: -1,3-linked glucans were predominantly synthesized when 1 mM UDP[¹⁴C]glucose was supplied; -1,4-linked glucans were predominantly synthesized when 1 mM [¹⁴C]-glucose was supplied. Thus the composition of the regenerated cell walls formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-³H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5-diphospho-D-mannose (GDP-mannose; [U-¹⁴C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.Abbreviations GlcNAc N-acetyl-D-glucosamine - GDP guanosine 5-diphospho - IEF isoelectric focusing - PHA phytohemagglutinin - SDS sodium dodecylsulfate - UDP uridine-5-diphospho     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

Inter-conversion of free sugars and accumulation of galactomannan in response to supplied metabolic mediators during pod filling in guar

Detached inflorescences of guar (Cyamopsis tetragonoloba), each bearing 4 uniformly-developing pods at 42 days post anthesis (DPA), were cultured for 6 days in complete liquid medium manipulated with a fixed concentration of mannose and varying concentration of myo-inositol. Such inflorescences, but with 2 pods, were also maintained in the solutions of (i) glucose(U-14C) containing myo-inositol or phytohormones, and (ii) mannose(U-14C) containing galactose for 36 hr. Effect of such exogenously supplied metabolic mediators on interconversion of free sugars in pod wall, endosperm and cotyledons and galactomannan accumulation in endosperm was studied. Myo-inositol decreased, over control, the relative proportion of invert sugars in pod wall, endosperm and cotyledons and at lower concentration (27.75 mM) it decreased the level of free sugars in pod wall and galactomannan in endosperm. In all pod tissues, 14C from both glucose and mannose got incorporated into myo-inositol as well as various sugars and maximum incorporation occurred in sucrose. High concentration of total free sugars and their 14C activity in pod wall indicated that this pod tissue was a potent accumulator of free sugars. With myoinositol, the relative proportion of 14C from glucose into raffinose sugars of pod wall and endosperm increased with a simultaneous decrease in this incorporation into galactomannan of the latter. Accompanying this, relative proportion of 14C into hexoses and myo-inositol decreased in pod tissues. Galactose increased 14C incorporation from mannose into total free sugars, sucrose and galactomannan with a concomitant decline in the labelling of hexoses. IAA and ABA enhanced 14C incorporation from glucose into total free sugars and this enhancement was much higher with IAA than ABA. The latter inhibited 14C incorporation into galactomannan. Based on these results, it was suggested that myo-inositol at lower concentration was inadequate to mediate the metabolism of sugars and, thereby, galactomannan synthesis. Galactose and mannose exhibited a mutual beneficial effect on their transportation to pods. Phytohormones stimulated the accumulation of sucrose in pod wall for its obligatory unloading into the seed.     

Biosynthesis of legume-seed galactomannans in vitro

Particulate enzyme preparations were isolated from developing fenugreek (Trigonella foenum-graecum L.) and guar (Cyamopsis tetragonoloba [L.] Taub.) seed endosperms during the period of galactomannan deposition in vivo. These preparations catalysed the formation of polysacharide products from guanosine 5-diphosphate (GDP)-mannose, from uridine 5-diphosphate (UDP)-galactose and from mixtures of the two nucleotides. The products were analysed by solubility, by complete acid hydrolysis, and by selective enzymatic cleavage using pure enzymes of known specificity. With GDP-[U-¹⁴C]-d-mannose as substrate and a divalent metal cation (Mg⁺², Mn⁺², or Ca⁺²) a highly efficient transfer of labelled d-mannosyl residues was obtained to give a product identified as linear (14)--linked d-mannan. No transfer of galactosyl residues was obtained when GDP-[U-¹⁴C]-d-galactose was the only substrate, although very low and variable amounts of an unidentified product which released labelled glucose on acid hydrolysis were formed. In the presence of UDP-galactose, GDP-mannose and Mn⁺² ions, products were formed which have been characterised as galactomanans — a linear (14)--d-mannan backbone carrying d-galactopyranosyl substituents linked (16)- to mannose. The degree of galactose substitution of the d-mannan backbone was manipulated in vitro by varying GDP-mannose concentrations at constant (saturating) UDP-galactose levels. The transfer of d-galactosyl residues from UDP-galactose to galactomannan was absolutely dependent upon the simultaneous transfer of D-mannosyl residues from GDP-mannose. d-Mannan sequences pre-formed in situ using the mannosyltransferase in the absence of UDP-galactose could not become galactose-substituted in a subsequent incubation either with UDP-galactose alone or with UDP-galactose plus GDP-mannose A model for the interaction of GDP-mannose mannosyltransferase and UDP-galactose galactosyltransferase in galactomannan biosynthesis is proposed.Abbreviations GDP guanosine 5-diphosphate - TLC thinlayer chromatography - UDP uridine 5-diphosphate