Purification of apyrase from yellow lupin cotyledons after extraction with perchloric acid.

Neutralized 1 M perchloric acid (PCA) extracts of yellow lupin (Lupinus luteus) seedling cotyledons contain considerable amounts of apyrase (EC 3.6.1.5). Investigators who use PCA extraction for the estimation of nucleotide levels, particularly in plant tissues, should be aware of this danger. Only when the material is treated with 1.8-2 M PCA are the extracts obtained free of apyrase activity. Chromatography of neutralized 1 M extracts obtained from 7-day-old seedling cotyledons on DEAE-Sephacel and Sephadex G-100 yields almost homogeneous apyrase that shows a band of M(r) 51,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The molecular weight of the native enzyme is also about 51,000. The apyrase preparation is free of nonspecific phosphatases, nucleotidases, and adenosine nucleosidase, as well as dinucleoside polyphosphate-degrading enzymes. The apyrase exhibits a broad pH optimum between 6 and 8. Mg2+ and Ca2+ are required for maximum activity; Zn2+ and Mn2+ are less effective and Co2+, Ni2+, and Cd2+ are without effect. The Km values for ATP and ADP are about 20 microM. All common 5'-nucleoside tri- and diphosphates as well as adenosine 5'-tetraphosphate are substrates.     

Purine catabolism in plants : purification and some properties of inosine nucleosidase from yellow lupin (lupinus luteus L.) seeds

Inosine nucleosidase (EC 3.2.2.2), the enzyme which hydrolyzes inosine to hypoxanthine and ribose, has been partially purified from Lupinus luteus L. cv. Topaz seeds by extraction of the seed meal with low ionic strength buffer, ammonium sulfate fractionation, and chromatography on aminohexyl-Sepharose, Sephadex G-100, and hydroxyapatite.

Molecular weight of the native enzyme is 62,000 as judged by gel filtration. The inosine nucleosidase exhibits optimum activity around pH 8. Energy of activation for inosine hydrolysis estimated from Arrhenius plot is 14.2 kilocalories per mole. The K_m value computed for inosine is 65 micromolar.

Among the inosine analogs tested, the following nucleosides are substrates for the lupin inosine nucleosidase: xanthosine, purine riboside (nebularine), 6-mercaptopurine riboside, 8-azainosine, adenosine, and guanosine. The ratio of the velocities measured at 500 micromolar concentration of inosine, adenosine, and guanosine was 100:11:1, respectively. Specificity (V_max/K_m) towards adenosine is 48 times lower than that towards inosine.

In contrast to the adenosine nucleosidase activity which is absent from lupin seeds and appears in the cotyledons during germination (Guranowski, Pawełkiewicz 1978 Planta 139: 245-247), the inosine nucleosidase is present in both lupin seeds and seedlings.

     

Diverse regulation by sucrose of enzymes involved in storage lipid breakdown in germinating lupin seeds

The regulatory function of sucrose in the activity of lipid-degrading enzymes was investigated in germinating seeds of yellow lupin (Lupinus luteus L.), white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet). The study was conducted on isolated embryo axes, excised cotyledons and seedlings cultured in vitro for 96 h on medium with 60 mM sucrose or without the sugar. The activity of lipase (lipolysis), acyl-CoA oxidase and catalase (fatty acid β-oxidation) was enhanced in all studied organs cultured on medium without sucrose. The activity of cytosolic aconitase (glyoxylate cycle) was stimulated by sucrose in seedling axes and isolated embryo axes, whereas in seedling cotyledons and excised cotyledons, it was inhibited. The regulatory function of sucrose in phosphoenolpyruvate carboxykinase (gluconeogenesis) was observed only in isolated embryo axes and the activity was lower in carbohydrate deficiency conditions. The peculiar features of storage lipid breakdown in germinating lupin seeds and its regulation by sucrose are discussed.     

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus)

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.     

Response of seeds ofLupinus termis andVicia faba to the interactive effect of salinity and ascorbic acid or pyridoxine

The interactive effect of salinity and presoaking in ascorbic acid or phyridoxine on germination, seedling growth, and some relevant metabolic changes ofLupinus termis andVicia faba seeds were studied. Germination studies indicated that broad bean tolerated NaCl salinity up to 240mM NaCl and lupin to 200mM NaCl. The lengths of roots and shoots and their water content, as well as dry matter yield, remained more or less unchanged up to the level of 80mM NaCl. Salinity induced marked progressive increases of carbohydrates and proline in broad bean and soluble protein in lupin seedlings, irrespective of the salinity level used. The other organic solutes (soluble protein in broad bean and carbohydrates in lupin seedlings) remained more or less unchanged at low and moderate levels of NaCl. However, under the higher salinity levels, in lupin the losses in carbohydrates were accompanied by increases in soluble protein, whereas in broad bean an opposite effect was obtained. The level of 40mM NaCl had a pronounced stimulatory effect on the all the variables studied. Presoaking seeds in either ascorbic acid or pyridoxine counteracted the adverse effects of salinity on germination and seedling growth as well as on some metabolic mechanisms of lupin and broad bean plants. The importance of these processes to the salinity tolerance of broad bean and lupin have been discussed.     

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.     

Redistribution of Nutrient Elements from Cotyledons of Two Species of Annual Legumes During Germination and Seedling Growth

The distribution of dry matter and various mineral nutrientsbetween testa and embryo of seeds of Lupinus albus and L. angustifoliusis described It was found that lupin seeds at either end ofa pod contained less dry matter and minerals than seeds in themiddle of the fruit. The transport of dry matter, N, P, K, S,Ca, Mg, Na, Fe, Zn, Mn and Cu from cotyledons of parent seedsof both species to the seedling axis was measured from germinationto the time of cotyledon death. N, P, K and S were retrievedfrom cotyledons with over 90 per cent efficiency, dry matter,Mg, Na, Fe, Zn, Mn and with 59–90 per cent efficiency,and Ca with 26–31 per cent efficiency. There was littlechange in the efficiency of nutrient retrieval from cotyledonswhen seedlings were grown in different culture media. Both speciesshowed a linear relationship between the loss of each elementand dry matter throughout the experiment, and a similar proportioningbetween root and shoot of the amount of a specific nutrientmobilized from cotyledons of parent seeds. Lupinus albus L., Lupinus angustifolius L., lupin, transport, of dry matter and mineral nutrients     

Asparagine Enhances Starch Accumulation in Developing and Germinating Lupin Seeds

Regulation of starch accumulation in yellow (Lupinus luteus L.), white (L. albus L.), and Andean lupin (L. mutabilis Sweet) developing and germinating seeds was investigated. Research was conducted on cotyledons isolated from developing seeds as well as on organs of germinating seeds, that is, isolated embryo axes, excised cotyledons, and seedling axes and cotyledons. All organs were cultured in vitro for 96 h in different carbon (60 mM sucrose) and nitrogen (35 mM asparagine or 35 mM nitrate) conditions. Ultrastructure observation showed one common pattern of changes in the number and size of starch granules caused by sucrose, asparagine, and nitrate in both developing and germinating seeds. Sucrose increased the number and size of starch granules. Asparagine additionally increased starch accumulation (irrespective of sucrose nutrition) but nitrate had no effect on starch accumulation. Asparagine treatment resulted in a significant decrease in soluble sugar level in all organs of germinating lupin seeds of the three species investigated. The above-mentioned changes were most clearly visible in white lupin organs. In white lupin, starch granules were visible even in cells of sucrose-starved isolated embryo axes where advanced autophagy occurs. The importance of asparagine-increased starch content in the creation of a strong source–sink gradient in developing and germinating lupin seeds is discussed.     

Biochemical and immunocytochemical localization of a BAPAase in developing and mature lupin cotyledons

Summary Activity measurements and specific antibodies were used to detect and localize in developing and mature cotyledons ofLupinus albus seeds an endopeptidase, active on BAPA, previously isolated from the same seeds. Total activity and enzyme amount were highest at full seed maturation and then declined during germination. Protein bodies were isolated from mature dry cotyledons under anhydrous conditions with a yield of intact organelles of about 80% as assessed by dot blotting with antibodies to lupin legumin-like storage globulin. Activity assays on the isolated protein bodies indicated that 72% of BAPAase activity was associated with these organelles. Quantitative immunocytolocalization with antibodies to the enzyme on thin sections of mature lupin cotyledons confirmed that 75% of the enzyme was located inside the protein bodies. The possible involvement of the BAPAase in the proteolytic processing of the storage proteins during seed ontogeny is discussed.Abbreviations BAPA N-benzoyl-L-arginine-4-p-nitroanilide - DAF days after flowering - EM electron microscopy - NaPi sodium phosphate buffer - LRW London resin white - SDS sodium dodecylsulphate - PAGE polyacrylamide gel electrophoresis     

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.     

Photosynthesis, reserve mobilization and enzymes of sucrose metabolism in soybean (Glycine max) cotyledons

Soybean (Glycine max L. [Merr] cv. Ransom II) seedlings were grown under a light/ dark regime or in continuous darkness. Cotyledons were harvested daily for measurements of reserve mobilization, net carbon exchange rate, chlorophyll content and activities of certain enzymes involved in sucrose metabolism. Seedlings lost dry weight for the first 3 to 4 days after planting, then maintained a constant dry weight in the etiolated seedlings, and gained dry weight (via net fixation of CO₂) in the light-grown seedlings. In general, the patterns of reserve mobilization were as expected based on the collective work of other investigators. Soluble sugars were mobilized first, followed by protein and lipid. Galactinol, previously uncharacterized in soybean cotyledons, was present at low concentrations and was rapidly depleted within 2 days after planting. Mobilization of reserves was most important during the first 8 days after planting, whereas net cotyledonary photosynthesis began at 6 days after planting and was the primary source of assimilates after 8 days. Maximum rates of cotyledon photosynthesis were higher [up to 18 mg CO₂ (g dry weight)^?1 h^?1] than previously reported and accounted for about 75% of the assimilates transported from the cotyledons to the growing seedling during the functional life of the cotyledon. Enzyme activities in light-grown cotyledons peaked 7 to 10 days after planting and then declined. Sucrose phosphate synthase (EC 2.4.1.14) and sucrose synthase (EC 2.4.1.13) activities were similar in etiolated and light-grown seedlings, whereas uridine-5′-di-phosphatase (EC 3.6.1.6) activity was substantially higher in light-grown seedlings. During the period of reserve mobilization, the maximum sucrose phosphate synthase activity in cotyledonary extracts was in excess of the calculated rate of sucrose formation. However, when the cotyledons had highest net photosynthetic rates (14 days after planting), sucrose phosphate synthase activity was similar to the rate of carbon assimilation. It appears that soybean cotyledons are adapted for high rates of sucrose formation (from reserve mobilization and/or photosynthesis) for export to the rapidly growing tissues of the seedling.     

ACC conversion to ethylene by sunflower seeds in relation to maturation,germination and thermodormancy

Conversion of exogenous 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene was studied in sunflower (Helianthus annuus L., cv. Mirasol) seeds in relation to germinability. Ethylene production from ACC decreased during seed maturation, and non-dormant mature seeds were practically unable to synthesize ethylene until germination and growth occurred, indicating that ethylene forming enzyme (EFE) activity developed during tissue imbibition and growth. ACC conversion to ethylene was reduced by the presence of pericarp, and in young seedlings it was less in cotyledons than in growing axes.ACC conversion to ethylene by cotyledons from young seedlings was optimal at c. 30°C, and was strongly inhibited at 45°C. Pretreatment of imbibed seeds at high temperature (45°C) induced a thermodormancy and a progressive decrease in EFE activity.Abscisic acid and methyl-jasmonate, two growth regulators which inhibit seed germination and seedling growth, and cycloheximide were also shown to inhibit ACC conversion to ethylene by cotyledons of 3-day-old seedlings and by inbibed seeds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - CH cycloheximide - EFE ethylene forming enzyme - IAA indole-3-acetic acid - Me-Ja methyl-jasmonate     

A development-dependent hemagglutinin from cucumber surfaces: I. Hemagglutinin activity of cotyledon surfaces

During studies on the chemistry of plant surfaces, we observed that concomitant with the development of cucumber (Cucumis sativus L. cv `ELEM') cotyledons an agglutinin that agglutinates human erythrocytes appeared on epicuticular surfaces. The agglutinin was released from cotyledon surfaces into distilled water by a 1-minute immersion (or even less). Homogenization of the washed cotyledons released residual agglutinating activity. The surface-located hemagglutinating activity was age-dependent and occurred in dark- and light-grown cotyledons. Agglutinating activities were present in light-grown cotyledons of 2- to 14-day-old seedlings and in dark-grown cotyledons of 3- to 17-day-old seedlings. Agglutinating activities were maximal in cotyledons of 3- to 4-day-old seedlings. No activity could be detected in dry seeds or in seedlings up to 2 days and after 17 days of germination. The hemagglutinating activity was specifically inhibited by N,N′,N″-triacetylchitotriose, chitin, and chitosan.     

THE EFFECT OF COTYLEDON EXCISION ON THE GROWTH OF PEA SEEDLINGS

Pea plants grown under controlled conditions for 28 days were sampled nine times during this growth period. On each sampling day 80 plants were harvested. Cotyledons were excised from 40 plants and the root-shoot axis allowed to continue growth. The other 40 plants were divided into roots, shoots, and cotyledons; the dry weights of alcohol-water extracts and residues from these organs were determined. Shoot lengths, numbers of nodes, buds, flowers, and fruits of 28-day-old plants, whose cotyledons were excised at different times, were determined. These data indicate that cotyledon excision after the 13th day has no apparent effect on seedling growth, whereas excision before the 13th day reduces growth. Glucose-U-C¹⁴ supplied to attached cotyledons of 6- and 16-day-old seedlings resulted in no major difference in the distribution pattern of recovered label throughout the plant even though 16-day-old cotyledons were depleted of their normal endogenous food reserve.     

Activation of isoflavone biosynthesis in excised cotyledons of Lupinus seedlings by jasmonoids and excess light

Exogenous jasmonic acid (JA) and methyl jasmonate (MJ) induced accumulation of isoflavone constituents in cotyledons prepared from imbibed seeds of white lupin (Lupinus albus L.). Exogenous 0.2 mM MJ enhanced the levels of 7-O-(6"-O-malonyl)glucosylgenistein and 7-O-glucosylgenistein in the cotyledons of etiolated seedlings that had been incubated in the dark for 48 h. Regarding isoflavone induced by excision and slicing in the cotyledons as background level, the effect of light was 2- to 3-fold higher than that of 0.2 mM MJ. Cotyledons exposed to MJ along with a 24-h light period displayed a higher level of isoflavone accumulation than that of light alone. Total molar amounts of isoflavone accumulated in the cotyledons treated with MJ under continuous light were approximately the sum of those induced by MJ alone and light alone, respectively. The additive-like effect of MJ and light on isoflavone accumulation in lupin tissues suggested the presence of two different signaling systems independently responsible for those two stimuli. Excised cotyledons from etiolated yellow lupin (L. luteus L. cv. Topaz) seedlings also supported this hypothesis. The cotyledons could accumulate both an isoflavone and a flavone, and MJ selectively increased some of the isoflavone constituents, whereas light enhanced the levels of both. The selective accumulation mechanism of isoflavonoids in cotyledons, in which jasmonoids are involved, clearly differed from that activated by light.     

Changes in the activity of phosphoenolpyruvate carboxylating enzymes in germinating yellow lupin seeds

The rate of phosphoenolpyruvate carboxylation by extracts from germinating lupin seeds was measured through the H¹⁴CO₃ fixation. PEP carboxylation in seed axes increased during their imbibition, mainly as a result of the increase in the activity of PEP carboxylase [EC 4.1.1.31]. However, the activity of PEP carboxykinase [EC 4.1.1.38], present during the first 3 hours of imbibition, as well as the activity of PEP-carboxykinase [EC 4.1.1.49], after 24 hours of imbibition, have also been shown. Possible physiological role of the changes in the activity of PEP carboxylases during lupin seeds germination is discussed.     

Impacts of secondary seed dispersal and herbivory on seedling survival in Aesculus turbinata

Abstract. Aesculus turbinata is a tree species with large seeds (6.2 g mean dry weight). We studied the demography of its seeds and seedlings in a temperate deciduous forest in northern Japan to elucidate the ecological significance of large seeds with special reference to herbivory and secondary dispersal. Both seed and seedling stages suffered greatly from herbivores. Seedling herbivory was important judged from experiments with shoot clipping and hypogeal cotyledon removal. However, some seedlings survived through re-sprouting after herbivory. Survival rate and percentage resprouting seedlings were lower than those with remaining cotyledons, though seedling size was not affected. This suggests that stored resources in hypogeal cotyledons are working as a kind of ‘risk hedge’ against severe aboveground shoot clipping experienced by A. turbinata. The spatial distribution of seedlings was expanded via seed scatter-hoarding by rodents. Seedling survival rate was higher within canopy gaps than under closed canopy, indicating that canopy gaps are safe sites for establishment, and was negatively correlated with seedling density. Therefore, secondary seed dispersal in this species seems to be effective in ‘finding’ safe sites and in ‘escaping’ density-dependent mortality. The large seeds and seedlings of A. turbinata are attractive to herbivores, but the high resistance of seedlings to herbivory due to large reserves and the effective secondary dispersal appear to mitigate these disadvantages.     

The distribution of phospholipase D in developing and mature plants

1. The distribution of phospholipase D (phosphatidylcholine phosphatido-hydrolase, EC 3.1.4.4) was examined in the tissues of a number of plants and seeds. 2. The highest activities were found in various swollen storage tissues of certain plants: cabbage, central stalk; cauliflower, flower; celery, swollen leaf stalk; Kohl rabi, swollen stem; carrot, root; pea and marrow, seed. 3. Appreciable activity was retained in pea seeds for at least 1 year after drying. After germination and growth in the dark the total activity present in the cotyledons and also in the whole seedling decreased. 4. In the growing pea seedling (7 days old), about 3% of the total activity was in the plumule, 9% in the root and the remainder in the cotyledons. However, the activity in the root on a dry-weight basis was higher than that in the cotyledons. In both the root and the plumule the activity on a wet- or a dry-weight basis was highest in the growing tip. 5. The activity per dry weight in the roots and aerial parts of pea plants declined to low values as growth continued, but roots struck from cuttings of mature plants showed the same high activity as found in roots from young seedlings with cotyledons attached. 6. The total phospholipids present in the cotyledons of pea seeds were depleted on germination and growth. Of the individual phospholipids, phosphatidylcholine and phosphatidylethanolamine showed the same loss in 11 days as the whole phospholipid fraction, whereas phosphatidylinositol was decreased to a greater extent and cardiolipin and phosphatidylserine were not decreased. There was no increase of phosphatidic acid, as might have been expected if the phospholipids had disappeared through phospholipase D hydrolysis. 7. It is concluded that phospholipase D in plant storage tissues and seeds may be related to the rapid growth involved in their formation rather than being necessary for the utilization of their food reserve substances.     

Distribution of ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus

Kaurene synthetases catalyse the biosynthesis of ent-kaurene, a precursor of the gibberellins. In 4-day-old dark- or light-grown Helianthus annuus seedlings, the cotyledons contained over 90% of the synthetase activity. The low enzyme activity in the seedling hypocotyls and roots is not a consequence of inhibitory factors in these tissues. The cotyledons not only have the highest kaurene synthetase activity, but also have the highest inhibitory activity. The differences in kaurene synthetase activities in the different tissues cannot be explained on the basis of the levels of inhibitor(s) in the extracts. The mature perennial root of Marah macrocarpus has very low kaurene synthetase activity, in contrast to the liquid endosperm of immature seeds of the same plant which is a rich source of the enzyme.