Galactomannan from the seeds of chinese honey locust (Gleditsia sinensis Lam.)

By the hot water extraction method, galactomannan was extracted (4.5% yield of the seed mass) from the seeds of Chinese honey locust (Gleditsia sinensis Lam). It had a molecular weight of 1230 kDa, and its solutions had a high viscosity [η] of 1064 ml/g and optical activity [α]_D of +21.4°. The polysaccharide consists of mannose and galactose residues in the molar ratio 2.69: 1. In the galactomannan macromolecule the backbone is formed by 1,4-β-D-mannopyranose residues, 37% of which are substituted by α-D-galactopyranose at C6. By ¹³C-NMR-spectroscopy, fragments of differently galactose-substituted mannobiose units were found to be in the galactomannan being studied: Man-Man, (Gal)Man-Man, and Man-Man(Gal) in the ratio of 0.23: 0.47: 0.30.     

α-d-Galactosidase activity and galactomannan and galactosylsucrose oligosaccharide depletion in germinating legume seeds

Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH₄)₂SO₄ fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.     

Lipid biosynthesis in developing and germinating soybean cotyledons: The formation of palmitate and stearate by chopped tissue and supernatant preparations

Chopped tissue from developing soybean cotyledons incorporated [1-¹⁴C]acetate into palmitate, stearate, oleate, and linoleate, but with germinating cotyledons much less [1-¹⁴C]acetate was incorporated and the principal labeled products were palmitate, stearate, and oleate. When supernatant fractions from developing cotyledons were incubated with [1-¹⁴C]acetate or [2-¹⁴C]malonate the principal labeled products were palmitate and stearate. Supernatant fractions from germinating seed incorporated [2-¹⁴C]malonate into palmitate and also into short chain fatty acids including decanoate, laurate, and myristate. Supernatants from developing cotyledons required acyl carrier protein (ACP), ATP, CoA, and reduced pyridine nucleotides for maximal rates of incorporation of either [1-¹⁴C]acetate or [2-¹⁴C]malonate into palmitate and stearate. The de novo fatty acid synthetase which converts acetyl- and malonyl-ACP's to palmityl ACP was active in supernatant fractions from both young and old developing cotyledons. The elongation system, converting palmityl ACP to stearyl ACP, was more active in supernatants from younger than from older developing cotyledons. In experiments with chopped tissue the elongation system appeared equally active throughout the development process. These results are consistent with the view that the de novo and elongation systems are separate entities and that the elongation system in older cotyledons is less stable to the methods used to prepare supernatant fractions.     

Import of Sucrose and its Partitioning in the Synthesis of Galactomannan and Raffinose-oligosaccharides in the Developing Guar (Cyamopsis tetragonolobus) Seed

Import of sucrose and its transformation to galactomannan andraffinose-oligosaccharides have been studied in the developingguar seed. The amount of galactomannan gradually increased withthe ageing of the seed. During the entire period of pod development,sucrose constituted the major portion of the free sugars inthe seed (both endosperm and cotyledons) as well as in the podwall. Besides myo-inositol, the free sugars detected in thedeveloping endosperm and cotyledons were glucose, fructose,raffinose and stachyose. Some compounds, possibly glycosides(RG values higher than that of fructose), were also detectedin the endosperm. In the later stages of seed development, therelative proportion of raffinose in the free sugars increased,reaching 50% of the total free sugars in 77-d-old cotyledons.With pod maturity, the activities of soluble acid and boundacid invertases in the pod wall increased manifold with a concomitantdecline in the non-reducing sugar content. These enzymes seemto be involved in the mobilization of sucrose from this fruitingstructure into the seed. An increased synthesis of raffinose-oligosaccharidesboth in the endosperm and cotyledons was associated with highactivities of soluble acid invertase (pH 4.8) and sucrose-UDPglucosyl transferase in these tissues. Feeding uniformly labelled¹⁴C-sugars to the detached intact pods as well as to the isolatedendosperm and cotyledons resulted in labelling of all endogenousfree sugars and galactomannan. The uptake and incorporationinto galactomannan of ¹⁴C was stimulated by Co²⁺, Mn²⁺ and Mg²⁺.Except for mannose, a major proportion of the ¹⁴C from glucose,fructose and sucrose appeared in sucrose in both endosperm andcotyledons indicating a fast reconstitution of sucrose in situ.Based on the present results, a possible mode of transformationof sucrose to galactomannan and raffinose-oligosaccharides hasbeen proposed. Key words: Sucrose, galactomannan, raffinose-oligosaccharides, invertase, sucrose-UDP glucosyl transferase, ¹⁴C-incorporation, guar seed     

Galactomannan changes in developing Gleditsia triacanthos seeds

Galactomannan has been extracted from the endosperm of seeds of Gleditsia triacanthos (honey locust) at different stages of development, when the seed was accumulating storage material. Properties of the different samples have been studied. The molecular size distribution became more disperse as galactomannan accumulated and the galactose: mannose ratio decreased slightly. Some possible reasons for these changes are discussed.     

Interconversion and translocation of free sugars during galactomannan utilization in germinating guar (Cyamopsis tetragonoloba) seed

With the onset of the degradation of galactomannan, the galactose and mannose levels increased in the endosperm. The hydrolysis of galactomannan was more or less complete within the first 3 days of germination. In the cotyledons, sucrose was the predominant free sugar during the period of rapid galactomannan hydrolysis and reducing sugars (glucose + fructose) were present in only 10–20% proportion. The level of soluble acid invertase activity was in the order of embryonic axis > endosperm > cotyledons. On the basis of (a) absence of galactose and mannose, (b) high proportion of sucrose, (c) very fast conversion of [¹⁴C]glucose and [¹⁴C]mannose to [¹⁴C]sucrose and (d) very low levels of both soluble and bound invertases in cotyledons, we conclude that there is an active synthesis of sucrose in this tissue where disaccharide seems to be least hydrolysed during the period of galactomannan mobilization. A rapid hydrolysis of galactomannan in endosperm during early germination resulted in the synthesis of some starch, as a temporary reserve, in cotyledons. When the cotyledons entered the phase of first leaf formation, cotyledonary sucrose was hydrolysed giving rise to invert sugars. In the embryonic axis, the increase in the ratio of reducing sugars to sucrose coupled with a higher level of invertase, compared with sucrose-UDP glucosyl transferase, indicated that free sugars from the cotyledons are translocated to the embryonic axis as sucrose.     

Arginine catabolism in the cotyledons of developing and germinating pea seeds

Arginine is the predominant free amino acid in the cotyledons of developing seeds of Pisum sativum L. cv Marzia. Breakdown of arginine was measured by injecting l-[guanido-¹⁴C]arginine into detached cotyledons. Cotyledons of developing seeds showed a low rate of ¹⁴CO₂ evolution whereas a much higher rate of ¹⁴CO₂ evolution was measured from cotyledons of seeds 4 days after the onset of germination. The activities of the catabolic enzymes arginase, urease, and ornithine aminotransferase were measured throughout development and germination. Arginase and ornithine aminotransferase were present at an early stage of development. Urease activity appeared later as the seeds started to desiccate. During germination, all three enzymes were present. The different course of activity of these enzymes indicates that they are controlled separately.     

β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism – A key factor for the hydrolysis of galactomannan substrates

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75–25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.     

A celluloytic complex from <Emphasis Type="Italic">Clostridium cellulovorans</Emphasis> consisting of mannanase B and endoglucanase E has synergistic effects on galactomannan degradation

In our previous study using a fluorescently labeled cohesin biomarker, we detected and identified a putative cellulosomal mannanase belonging to the glycosyl hydrolase family 26 from Clostridium cellulovorans in xylan-containing cultures. In this study, a mannanase gene, manB from C. cellulovorans, was expressed in Escherichia coli. The optimal pH of a purified enzyme was around pH 7.0 and the optimal temperature was 40°C. The purified mannanase B (ManB) showed high hydrolytic activity toward galactomannan. An assembly of ManB with mini-CbpA, which contains a carbohydrate-binding module that provides proximity to insoluble substrates, increased the activity toward galactomannan [locust bean gum (LBG) and guar gum] 1.7- and 2.0-fold over those without mini-CbpA. We tested the synergistic effects on galactomannan (LBG and guar gum) degradation using cellulosomal mannanase ManB with cellulosomal endoglucanase E, which was predicted to have mannanase activity in C. cellulovorans as a cellulolytic complex. When assembled with the mini-CbpA, the mixture of endoglucanase E (EngE) and ManB at a molar ratio of 1:2 showed the highest synergistic effect (2.4-fold) on LBG. The mixture at a ratio of 1:3 showed the highest synergistic effect (2.8-fold) on guar gum. These synergistic actions indicated that ManB assembled with mini-CbpA hydrolyzed insoluble galactomannan, which in turn promoted soluble galactomannan degradation by EngE.     

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.     

Characterization of the activity of fatty-acid epoxide hydrolase in seeds of castor bean (Ricinus communis L.)

Epoxide hydrolase (EC 3.3.2.3) activity was measured with [1-¹⁴C]cis-9,10-epoxystearic acid as the substrate. Homogenates were prepared from the endosperm tissue of germinating seeds of castor bean (Ricinus communis L. zanzibariensis). The activity of fatty-acid epoxide hydrolase was characterized with respect to dependence on time, amount of protein, pH and temperature. Analyses of enzyme distribution in endosperm, cotyledons, root and hypocotyl showed the highest total activity in the endosperm, less in the cotyledons and low activity in the root and hypocotyl. The specific activity was similar for cotyledons and endosperm. Analysis of the temporal expression of the enzyme in the endosperm during germination revealed high activity already in the imbibed seed. Activity was maximal between days four to six and then decreased at the end of one week. Subcellular fractionation of endosperm revealed a dual distribution of activity between the glyoxysomal and the cytosolic fractions.     

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from Cyamopsis tetragonoloba (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO ₃ ^? -groups was 48.05 ± 2.31%. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests—aIIa and aXa. The antithrombin activity (aIIa) was high (up to 65–87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

The enzymatic synthesis of amyrin glucoside

Label appeared in several cell fractions isolated from the cotyledons of pea seeds germinated for 48 hr with mevalonate-[2-¹⁴C]. The major radioactive metabolite in each fraction was amyrin. In a similar experiment, a fraction sedimenting between 1000 and 25 000 g and a microsomal pellet were labeled with 3H from mevalonate-[2-³H]. Each of these tritiated fractions on incubation with UDP-glucose-[U-¹⁴C] yielded CHCl₃-MeOH-soluble material bearing ¹⁴C and ³H. TLC of the extracts gave a compound chromatographically identical with a glucoside and bearing the two isotopes. Acid hydrolysis of this compound gave an ether-soluble material carrying ³H alone. On TLC it co-chromatographed with amyrin. Of the two tritiated cotyledon fractions, the microsomal pellet had the lower glucosyltransferase activity. The labeled amyrin residing in this fraction served as an acceptor for glucose from UDP-glucose in the presence of a glucosyltransferase from pea seedling axis tissue. In such a mixed preparation, the axis tissue transferase suffers a marked inhibition by the cotyledon preparation.     

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds

Phosphomannomutase and phosphoglucomutase in developing Cassia corymbosa seeds have been completely separated from each other and from glucose phosphate and mannose phosphate isomerases by a series of chromatographic procedures that included affinity elution chromatography. Some properties, including the K_m for d-mannose 1,6-biphosphate with phosphomannomutase, are described. The activities of phosphoglucomutase and phosphomannomutase in some other plant tissues are also compared. The significance of these enzymes and the pathway of galactomannan synthesis are discussed.     

Effect of gibberellic acid on sterol production in Corylus avellana seeds

Gibberellic acid-induced germination of hazel seeds was accompanied by little change in the sterol content of the cotyledons. Dormant and germinating cotyledons rapidly incorporated [2-¹⁴C]MVA into squalene which was slowly converted to sterols. Gibberellin treatment induced an increase in the incorporation of [2-¹⁴C]MVA into cotyledon esterified sterols. An increase in free sterols occurred in the germinating embryonic axes, with increased relative amounts of stigmasterol and campesterol in the free 4-desmethylsterols. Germination was accompanied by increased incorporation of [2-¹⁴C]MVA into free and esterified sterols in the embryonic axes.     

Regulation of α-galactosidase activity and the hydrolysis of galactomannan in the endosperm of the fenugreek (Trigonella foenum-graecum L.) seed

When endosperms were isolated from fenugreek seeds 5 h after sowing and incubated in a small volume of water, the development of α-galactosidase activity and the breakdown of the galactomannan storage polysaccharide were both inhibited relative to control endosperms incubated in larger volumes. The inhibition could be relieved by pre-washing the endosperms, and reimposed by the wash-liquors. If the endosperms were isolated 24 h after sowing, no inhibition was observed. Removal of the embryonic axis from germinating fenugreek seeds and from germinated seedlings also inhibited the development of α-galactosidase activity and galactomannan breakdown in the endosperms; the inhibition was more pronounced the earlier the axis was removed. Axis excision 5 h after sowing caused a delay in the onset of galactomannan breakdown and of the appearance of α-galactosidase activity in the endosperms. It also led to a decrease in the rates of galactomannan breakdown and α-galactosidase production. Axis excision 24 h after sowing caused only a slowing of the rates of galactomannan breakdown and α-galactosidase increase. The inhibition caused by axis removal at 5 h could be relieved partially by gibberellin (10^-4 M), benzyladenine (10^-5 M), mixtures of these and by the herbicide SAN 9789 [4-chloro-5-(methylamine)-2-(α,α,α-trifluoro-m-tolyl)-3-(2H)-pyridazinone]. These substances had no effect on the inhibition caused by axis-removal at 24 h. Excision of the cotyledons at 5 h-leaving the separated axis and the endosperm-also caused inhibition of galactomannan breakdown and α-galactosidase development. The results are consistent with the presence in the fenugreek seed endosperm of diffusible inhibitors of galactomannan mobilisation which are removed or inactivated during normal germination and early seedling development. They are also consistent with a role for the seedling axis in the control of galactomannan breakdown in the endosperm. Initially the axis appears to have a regulatory function (via gibberellins and/or cytokinins?) in determining the onset of α-galactosidase production in the endosperm. Thereafter its continued presence is necessary to ensure maximal rates of α-galactosidase production and galactomannan hydrolysis. The role of the axis may be initially to counteract the endogenous inhibitors in the endosperm and then to act as a sink for the galactomannan breakdown products released in the endosperm and taken up by the cotyledons.     

Agar/galactomannan gels applied to shoot regeneration from tobacco leaves

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum — a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm⁻³ each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm⁻³). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.     

Anticoagulant activity of low-molecular-weight sulfated derivatives of galactomannan from <Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis> (L.) seeds

Galactomannan from seeds of Cyamopsis tetragonoloba (L.) Taub. (guar) was depolymerized using immobilized enzymatic preparation celloviridin. A set of fragments whose molecular weights varied from 12.6 to 245.6 kDa was obtained. Sulfated derivatives of components of all fractions were synthesized, in which the content of HSO ₃ ^? groups was 48.05 ± 2.31%. All preparations exhibited anticoagulant activity, which was recorded in vitro in two tests—alla and aXa. The antithrombin activity (aIIa) was high (up to 65–87 U/mg) and did not depend on the molecular weight of a sulfated derivative; in the second test (aXa), the effect of molecular weight was observed. Biospecific electrophoresis allowed us to detect the ability of galactomannan sulfates to form complexes with protamine sulfate, a classic antidote to heparin.     

Comparative study of storage compound breakdown in germinating seeds of three lupine species

Storage lipid and protein breakdown in germinating seeds of yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupine (L. mutabilis Sweet) and regulatory function of sucrose were investigated. Less oil bodies were detected in organs of yellow lupine seeds, whereas the highest content of oil bodies was noticed in the Andean lupine seeds. Mature, air-dried yellow, white and Andean lupine seeds do not contain starch. Starch grains appear the earliest in white lupine seeds during imbibition. Sucrose deficiency in tissues enhances breakdown of storage lipid, protein and temporary starch in cotyledons. In sucrose starved embryo axes of all investigated lupine species, an increased level of vacuolization was noted. Interconnections between catabolism of storage protein and storage lipid in germinating lupine seeds were identified by applying ¹⁴C-acetate. To assess the importance of key processes in storage lipid breakdown NaF (inhibitor of glycolysis and gluconeogenesis), KCN, NaN₃ and SHAM (inhibitors of mitochondrial electron transport chain) and MSO (inhibitor of glutamine synthetase) were used. Radioactivity coming from ¹⁴C-acetate was released as ¹⁴CO₂ but mostly was incorporated into ethanol-soluble fraction of embryo axes and cotyledons. Respiratory inhibitors caused a significant decrease in ¹⁴CO₂ and ethanol fractions in all three lupine species studied. MSO stimulated release of ¹⁴CO₂ and radioactivity of ethanol fractions in yellow lupine organs fed with sucrose, but in Andean lupine MSO enhanced the production of ¹⁴CO₂ and radioactivity of ethanol fractions both in organs fed and not fed with sucrose. Different strategies of storage compound breakdown are proposed, depending on relative proportion in storage protein and lipid content in lupine seeds.     

The Biosynthesis of Cytokinins in Germinating Lupin Seeds

Imbibed intact seeds, and excised embryos and cotyledons ofyellow lupin (Lupinus luteus L. cv. Weiko III) have been incubatedwith [¹⁴C]-adenine to investigate cytokinin biosynthesis duringthe early stages of germination. Following incubation the tissueswere extracted and purified by solvent partition and chromatographyon cellulose phosphate, diethylaminoethyl cellulose and SephadexLH-20 columns. Using a variety of thin layer chromatographic,high performance liquid chromato-graphic and chemical procedures,incorporation of ¹⁴C into dihydrozeatin riboside and its nucleotidewas demonstrated in extracts of intact embryos, intact cotyledonsand excised embryos. However, radioactivity was not found associatedwith cytokinins in fractions derived from the isolated cotyledons.This is the first direct demonstration of cytokinin biosynthesisin germinating seeds and the results indicate that the capacityfor cytokinin biosynthesis is probably confined to the embryonicaxes. If this is so, the levels of [¹⁴CJ-dihydrozeatin ribosideassociated with intact embryo and intact cotyledon fractionsindicate that the synthesized cytokinin is transported to andaccumulates in the cotyledons. Key words: Lupinus luteus, cytokinin biosynthesis, seed germination