Initiation of the degradation of the soybean kunitz and bowman-birk trypsin inhibitors by a cysteine protease

Protease K1 activity initiates the degradation of the Kunitz soybean trypsin inhibitor (KSTI) during germination and early seedling growth. This enzyme was purified nearly 1300-fold from the cotyledons of 4-day-old soybean (Glycine max [L.] Merrill) seedlings. Protease K1 is a cysteine protease with a molecular weight of approximately 29,000. It cleaves the native form of KSTI, Ti^a, to Ti^a_m, the same modified form observed in vivo. In addition to attacking KSTI, protease K1 is also active toward the major Bowman-Birk soybean trypsin inhibitor, as well as the α, α′, and β subunits of soybean β-conglycinin. The properties and temporal variation of protease K1 during germination indicate that it is responsible for initiating the degradation of both KSTI and Bowman-Birk soybean trypsin inhibitor in the soybean cotyledon.     

Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins

The protease that degrades the beta subunit of the soybean (Glycine max (L.) Merrill) storage protein beta-conglycinin was purified from the cotyledons of seedlings grown for 12 days. The enzyme was named protease C2 because it is the second enzyme to cleave the beta-conglycinin storage protein, the first (protease C1) being one that degrades only the alpha' and alpha subunits of the storage protein to products similar in size and sequence to the remaining intact beta subunit. Protease C2 activity is not evident in vivo until 4 days after imbibition of the seed. The 31 kDa enzyme is a cysteine protease with a pH optimum with beta-conglycinin as substrate of 5.5. The action of protease C2 on native beta-conglycinin produces a set of large fragments (52-46 kDa in size) and small fragments (29-25 kDa). This is consistent with cleavage of all beta-conglycinin subunits at the region linking their N- and C-domains. Protease C2 also cleaves phaseolin, the Phaseolus vulgaris vicilin homologous to beta-conglycinin, to fragments in the 25-28 kDa range. N-Terminal sequences of isolated beta-conglycinin and phaseolin products show that protease C2 cleaves at a bond within a very mobile surface loop connecting the two compact structural domains of each subunit. The protease C2 cleavage specificity appears to be dictated by the substrate's three-dimensional structure rather than a specificity for a particular amino acid or sequence.     

Soybean seed protein electrophoresis profiles from 15 Asian countries or regions: Hypotheses on paths of dissemination of soybeans from China

Soybean [Glycine max (L.) Merr.] seed protein extracts from 1,603 accessions obtained from 15 Asian countries or regions (not including Japan) were analyzed for the presence of alleles of 2 proteins. Three alleles of the Kunitz. trypsin inhibitor orSBTI-A ₂ designated asTi ^a,Ti ^b andTi ^c are electrophoretically distinguishable from one another by their different Rf values of 0.79, 0.75 and 0.83, respectively. The Sp₁ seed protein or β-amylase has 2 alleles designatedSp1 ^a andSp ₁ ^b which are electrophoretically distinguishable from one another by their Rf values 0.36 and 0.42. About 94 percent of the soybean accessions had theTi ^a allele. Two accessions from Korea,P.I. 157440 andP.I. 196168, do not have theSBTI-A2 protein(ti). Two accessions, one from Pakistan and the other from Korea, were identified as having theTi ^c allele. Only the Korean and central Indian soybean populations have a high frequency for theTi ^b allele. Within Korea, the soybeans from those districts that lie closest to Japan have a high frequency for theTi ^b allele whereas the soybeans from those districts that lie closest to China have a low frequency for theTi ^b allele. TheTi ^b allele is not present in soybeans from the Philippines, Vietnam, Thailand, Malaysia, Burma, Nepal, Pakistan, and Afghanistan. Only 1 accession each from Taiwan and Indonesia have theTi ^b allele. TheSp ₁ ^a allele is not present in soybeans from Taiwan, Vietnam, Thailand, Malaysia, Indonesia, Burma, Pakistan and Afghanistan. The highest frequency for theSp1 ^a allele occurs in soybean germ plasm from northern India and Nepal. The soybeans from Asia (including Japan) were divided into 3 gene centers— primary, secondary, and tertiary—containing 7 germ plasm pools. Paths of dissemination of the soybean from China to the rest of Asia were developed based upon a combination of electrophoretic data and available historical, agronomic, and biogeographical literature.     

Rapid release of protease inhibitors from soybeans: immunochemical quantitation and parallels with lectins

Specific antisera were prepared against the Bowman-Birk trypsin inhibitor and four other trypsin inhibitors of low molecular weight isolated from soybeans (Glycine max L. cv. Tracy). These antisera were used to detect the presence and amount of the inhibitors in: (a) seeds and protein extracts of soybean meal; (b) seedlings; and (c) the water surrounding the seeds and roots of seedlings. Lectin activities in seeds, seedlings, and water were also determined at the same time as the protease inhibitor activities. By competitive inhibition of immunoprecipitation, the combined five low molecular weight protease inhibitors were found to constitute the following percentages of proteins (w/w): 6.3% in defatted soybean meal; 8.1% of the protein extracted from the meal by a buffer of pH 8.6; 8.3, 14.7, 15.2, 16.1, 17.2, and 18.9% of the protein in a lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, respectively; 8.2% in a lyophilisate of water in which roots of seedlings grew for 20 days; 1.5% in cotyledons; and less than 0.1% in epicotyls, hypocotyls, and roots of 12-day-old seedlings. Hemagglutination activities, expressed as the lowest amount of protein required to give a positive agglutination of 0.2 ml of 2% rabbit red blood cells, were as follows: purified soybean lectin, 0.08 μg; lyophilisate of water in which seeds were incubated for 4, 8, 12, 16, 20, and 24 hours, 10, 2.5, 5, 5, and 2.5 μg, respectively; lyophilisate of water in which roots grew for 20 days, 5 μg; 12-day-old cotyledons, roots, epicotyls, and hypocotyls, 12.5, 100, >1,000, and >500 μg, respectively. The results indicate that a large amount of protease inhibitors as well as lectins are released from seeds during the first 8 hours of imbibition. Neither lima bean trypsin inhibitor (mol wt, 10,000) nor Kunitz soybean trypsin inhibitor (mol wt, 21,500) showed competitive inhibition in tests with antisera against low molecular weight soybean protease inhibitors.     

Purification and characterization of protease III from Escherichia coli.

An endoproteolytic enzyme of Escherichia coli, designated protease III, has been purified about 9,600-fold to homogeneity with a 6% yield. The purified enzyme consists of a single polypeptide chain of Mr 110,000 and is most active at pH 7.4. Protease III is very sensitive to metal-chelating agents and reducing agents. The EDTA-inactivated enzyme can be reactivated by Zn2+, Co2+ or Mn2+. Protease III is devoid of activity toward aminopeptidase, carboxypeptidase, or esterase substrates but rapidly degrades small proteins. When fragments of beta-galactosidase are used as substrates for protease III, the enzyme preferentially degrades proteins with molecular weights of less than 7,000. Protease III cleaves the oxidized insulin B chain at two sites with an initial rapid cleavage at Tyr-Leu (16-17) and a second slower cut at Phe-Tyr (25-26).     

Validation of SSR markers linked to null kunitz tryspin inhibitor allele in Indian soybean [Glycine max (L.) Merr.] population

Kunitz trypsin inhibitor, a proteinaceous antinutritional factor present in soybean seeds, is responsible for inferior nutritional quality of raw soybean and incompletely processed soy products. The objective of the present investigation was to validate the SSR markers (Satt228 and Satt409) reported to be linked to Ti locus in an Indian soybean population generated from the cross between soybean cultivar LSb1 (TiTi) and PI542044 (titi). Parental polymorphism was surveyed using Satt409, Satt228 and 5 SSR markers in the neighbouring genomic region of Ti locus. A portion of the cotyledon of F₂ seeds was used for analyzing the presence or absence of kunitz trypsin inhibitor polypeptide electrophoretically while the remaining portion containing the embryo was used for raising the F₂ plants (104) for the development of mapping population. The SSR marker Satt228 reported to be tightly linked with Ti locus was not found to be polymorphic for the parents used in our study. Satt409 was found to be linked with Ti locus at 4.7 cM. Besides, a new marker Satt538 was found to be linked with Ti locus at a distance of 17.8 cM. Thus, the SSR marker Satt409 can be useful for Marker Assisted Selection for transferring titi allele in the background of Indian soybean genotypes.     

Purine synthesis and catabolism in soybean seedlings : the biogenesis of ureides

The ureides, allantoin and allantoic acid, are the major nitrogenous substances transported within the xylem of N₂-fixing soybeans (Glycine max L. Merr. cv Amsoy 71). The ureides accumulated in the cotyledons, roots and shoots of soybean seedlings inoculated with Rhizobium or grown in the presence of 10 millimolar nitrate. The patterns of activity for uricase and allantoinase, enzymes involved in ureide synthesis, were positively correlated with the accumulation of ureides in the roots and cotyledons. Allopurinol and azaserine inhibited ureide production in 3-day-old cotyledons while no inhibition was observed in the roots. Incubation of 4-day-old seedlings with [¹⁴C]serine indicated that in the cotyledons ureides arose via de novo synthesis of purines. The source of ureides in both 3- and 4-day-old roots was probably the cotyledons. The inhibition of ureide accumulation by allopurinol but not azaserine in 8-day-old cotyledons suggested that ureides in these older cotyledons arose via nucleotide breakdown. Incubation of 8-day-old plants with [¹⁴C]serine suggested that the roots had acquired the capability to synthesize ureides via de novo synthesis of purines. These data indicate that both de novo purine synthesis and nucleotide breakdown are involved in the production of ureides in young soybean seedlings.     

Soybean lectin and related proteins in seeds and roots of le and le soybean varieties

The localizations of soybean lectin (SBL) and antigenically related proteins in cotyledons and roots of lectin positive (Le⁺) and lectin negative (Le⁻) soybean cultivars were compared by light level immunocytochemistry using antibodies produced against the 120 kilodalton (kD) native seed lectin tetramer or its subunits. Lectin is present in the protein bodies of cotyledons cells as are two other seed proteins, the Kunitz trypsin inhibitor and the storage protein glycinin. Analysis of single seed extracts by immunoblotting of sodium dodecyl sulfate-polyacrylamide gels using the same antibodies, reveals up to 4 milligrams of the 30 kD seed lectin protein is present per seed in the Le⁺ varieties. There is no detectable lectin in the protein bodies of Le⁻ cotyledons as determined by immunocytochemistry and immunoblotting. Enzyme-linked immunosorbent assay confirmed this result to a sensitivity of less than 20 nanograms per seed. In contrast, the roots of both Le⁺ and Le⁻ plants bind the seed lectin antibody during immunocytochemistry, with fluorescence mainly localized in vacuole-like bodies in the epidermis. Root extracts contain a 33 kD polypeptide that binds anti-SBL antibody at an estimated minimal level of 20 nanograms per 4-day seedling, or 2.0 nanograms per primary root tip. This polypeptide is also present in the embryo axis and in leaves. The latter also contain a 26 kD species that binds seed lectin antibody. The 30 kD seed lectin subunit, however, is not detectable in roots or leaves.     

The Amyloid Precursor Protein/Protease Nexin 2 Kunitz Inhibitor Domain Is a Highly Specific Substrate of Mesotrypsin

The amyloid precursor protein (APP) is a ubiquitously expressed transmembrane adhesion protein and the progenitor of amyloid-β peptides. The major splice isoforms of APP expressed by most tissues contain a Kunitz protease inhibitor domain; secreted APP containing this domain is also known as protease nexin 2 and potently inhibits serine proteases, including trypsin and coagulation factors. The atypical human trypsin isoform mesotrypsin is resistant to inhibition by most protein protease inhibitors and cleaves some inhibitors at a substantially accelerated rate. Here, in a proteomic screen to identify potential physiological substrates of mesotrypsin, we find that APP/protease nexin 2 is selectively cleaved by mesotrypsin within the Kunitz protease inhibitor domain. In studies employing the recombinant Kunitz domain of APP (APPI), we show that mesotrypsin cleaves selectively at the Arg¹⁵-Ala¹⁶ reactive site bond, with kinetic constants approaching those of other proteases toward highly specific protein substrates. Finally, we show that cleavage of APPI compromises its inhibition of other serine proteases, including cationic trypsin and factor XIa, by 2 orders of magnitude. Because APP/protease nexin 2 and mesotrypsin are coexpressed in a number of tissues, we suggest that processing by mesotrypsin may ablate the protease inhibitory function of APP/protease nexin 2 in vivo and may also modulate other activities of APP/protease nexin 2 that involve the Kunitz domain.     

Purification and Characterization of a Thermostable Alkaline Protease from Alkalophilic Thermoactinomyces sp. H8682

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatograhies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25, 000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0.

Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu²⁺ and Hg²⁺. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37°C for 60 min. The optimum pH was pH 11.5–13.0 at 37°C and the optimum temperature was 70°C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl₂, it showed maximum proteolytic activity at 80°C and stability from pH 4–12.5 at 60°C and below 75°C at pH 11.5. The stabilization by Ca²⁺ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of Microbiol serine protease, although alanine of the NH₂-terminal amino acid was deleted.     

Differential expression of kunitz and bowman-birk soybean proteinase inhibitors in plant and callus tissue

Bowman-Birk soybean trypsin inhibitor (BBSTI) but not Kunitz soybean trypsin inhibitor (KSTI) was found in samples of undifferentiated and partially differentiated Amsoy 71 tissue culture callus. This suggests the differential metabolism of these two classes of proteinase inhibitors, whether the difference be in synthesis, in rates of degradation, or both. The differential metabolism of the proteinase inhibitors is also seen in the plant. Both BBSTI and KSTI were found in the hypocotyl, root, and epicotyl of the Amsoy 71 soybean seedling in addition to their expected presence in the cotyledons. Whereas the ratio of KSTI to BBSTI in the cotyledon was higher, the ratio of BBSTI to KSTI was higher in the extracotyledonary tissues of the seedling. The levels of both classes of proteinase inhibitors declined during seedling growth, except in the epicotyl and the proximal root. In both of these tissues, an increase in BBSTI, but not in KSTI content, expressed as milligrams inhibitor per plant part, occurred.     

Ultrastructural localization of glycinin and β-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

Summary -Conglycinin (7S globulin) and glycinin (11S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7S globulin could not be detected.     

Ultrastructural localization of glycinin and beta-conglycinin in Glycine max (soybean) cv. Maple Arrow by the immunogold method

beta-Conglycinin (7 S globulin) and glycinin (11 S globulin) are the major reserve proteins of soybean. They were localized by the protein A immunogold method in thin sections of Glycine max (soybean) cv. Maple Arrow. In cotyledons, both globulins were simultaneously present in all protein bodies. Statistical analysis of marking intensities indicated no correlation between globulin concentration and size of protein bodies. The immunogold method failed to detect either globulin in the embryonic axis and in cotyledons of four-day-old seedlings. Similar observations were made with cotyledons of two soy varieties lacking either the lectin or the Kunitz trypsin inhibitor. In another variety (T-102) lacking the lectin, the 7 S globulin could not be detected.     

Dissemination of soybeans (Glycine max): Seed protein electrophoresis profiles among Japanese cultivars

Seed protein extracts from 477 Japanese soybean cultivars were analyzed by polyaery lamide gel electrophoresis to determine the distribution of the alleles of the Ti (Ti ^a,Ti ^b,Ti ^c) and Sp₁ (Sp ₁ ^a,Sp ₁ ^b loci with respect to maturity group and district of adaptation of each cultivar. About 60 percent of the soybean cultivars had theTi ^a allele. The frequency of theTi ^b allele was found to be highest in the southeast district and lowest in the northeast district. TheTi ^c allele discovered in 6 cultivars was traced to two possible sources adapted in the Tohoku District. TheSp _l ^a, allele was found in 26 cultivars ranging from Maturity Group II through VIII. The summer season type cultivars adapted to the Kyushu District having the genotypeTi ^b Sp_l ^b probably played a major role in the peculiar accumulation of theTi ^ub allele and the decrease of theSp _l ^a allele in the Japanese soybean cultivar population.     

Glycinin A4A5 subunit digesting protease in soybean seeds.

Endopeptidase was partially purified from the globulin fraction of 4-hr-imbibed soybean seeds. The protease fraction obtained had proteolytic activity on the glycinin A4A5 subunit at both pH 4 and 8. A suitable peptidic substrate for the endopeptidase was isolated from the tryptic digest of the carboxymethylated A4A5 subunit. Using the tryptic peptide of glycinin A5 subunit, a simple assay system for the soybean endopeptidase activity has been established. The activity was significantly inhibited by phenylmethylsulfonyl fluoride, indicating the endopeptidase is a serine protease.     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Identification of a plant introduction soybean line with genetic lesions affecting two distinct glycinin subunits and evaluation of impacts on protein content and composition

Unlike other oilseeds, soybean (Glycine max [L.] Merr) is also valuable due to its direct conversion into human food. One notable example is the cheese-like product tofu. The quality of tofu is improved when protein subunits derived from two glycinin genes, Gy1 and Gy4, are reduced or absent. Here we report the discovery that one exotic soybean plant introduction line, PI 605781 B, has not only a previously described loss-of-expression mutation affecting one glycinin gene (gy4), but also bears an extremely rare, potentially unique, frameshift mutation in the Glycinin1 gene (gy1-a). We analyzed glycinin gene expression via qRT-PCR with mRNA from developing seeds, which revealed that the novel allele dramatically reduced Gy1 mRNA accumulation. Similarly, both A₄A₅B₃ and A_1aB_1a protein subunits were absent or at undetectable levels, as determined by two-dimensional protein fractionation. Despite the reduction in glycinin content, overall seed protein levels were unaffected. The novel gy1-a allele was found to be unique to PI 605871B in a sampling of 247 diverse germplasm lines drawn from a variety of geographic origins.     

Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the alpha'/alpha or the beta subunit of beta-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the alpha' and alpha subunits of beta-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the beta subunit of soybean beta-conglycinin, were not degraded by protease C1. Degradation of soybean beta-conglycinin involves a sequential attack of the alpha subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three beta-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the alpha subunit at regions bearing long strings of acidic amino acid residues.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

β-Galactosidase in Alkalophilic Bacillus

The changes in ¹³C-NMR and ³¹P-NMR spectra and ¹H-NMR images in soybean cotyledons during germination were investigated. Using ¹³C-NMR, fatty acid signals in the form of triglycerides were observed in dry seeds, and those were observed approximately 18 days after the start of imbibition. Sucrose signals appeared at 16 hr and disappeared at 5 days. A signal was observed after 5 days, suggesting the activation of membrane metabolism in the cotyledons.

³¹P-NMR signals appeared 2 hr after imbibition before any apparent change in the ¹³C-NMR spectrum. The peaks identified as sugar phosphate, inorganic phosphate in the cytoplasm and in the vacuole, and an unassigned compound, were distinguishable after 5 days. The vacuole-associated inorganic phosphate peak became prominent 18 days after imbibition in ³¹P-NMR.

Distribution maps of free water indicated that the stored macro-molecular materials which bound water were consumed heterogeneously within the cotyledon. The relaxation time (T₁) increased suddenly between 18 days and 23 days after imbibition, which indicates the consumption of stored materials.

These findings suggest that cotyledons are the source of such compounds and the energy required for plant growth for approximately 18 days from germination until tri-foliolate leaves begin developing.