Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA.     

A 20-kDa Protein with the GTP-Binding and Trypsin Inhibitory Activities from Glycine max

A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65% identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.     

山东产野生大豆胰蛋白酶抑制剂的初步研究

该实验建立了HPLC测定大豆胰蛋白酶抑制剂(STI)活性的方法,并对山东产野生大豆(G.soja)与同地区产的黑豆和黄豆(G.max)的胰蛋白酶抑制活性差异进行了比较.用耦合了胰蛋白酶的亲合色谱柱对野生大豆的STI进行分离纯化,紫外分光光度法比较3种大豆的STI含量;PCR结合TA克隆技术对野生大豆STI中的Kunitz型(KSTI)蛋白基因编码区的氨基酸顺序进行初步测定.结果发现,山东产野生大豆的STI活性和含量均高于同地区产的黑豆和黄豆;山东产野生大豆的KSTI蛋白基因编码区的氨基酸顺序与已知的Tia型基本一致,仅第59位氨基酸由于单核苷酸的置换发生了Ser→Thr的转变,此位置位于活性中心附近.研究表明,山东产野生大豆胰蛋白酶抑制活性较强,且含量高.     

Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterium tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (R₀) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues. Putative transformed R₀ soybean plants were advanced to produce R₁ plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the R₀ plant, and that about 1/10 of these plants yielded transformed R₁ plants. The presence of the Nos-NPT II gene in DNAs isolated from both R₀ and R₁ plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R₁ soybean plants.     

Flower and Pod Development in Water-Deficient Soybeans (Glycine max L. Merr.)

Water deficits during flowering decrease the number of seed-bearingpods in soybean (Glycine max L. Merr.). Failure to set podsmay indicate an inherent sensitivity to low tissue water potential(     

Dehydration Injury in Germinating Soybean (Glycine max L. Merr.) Seeds

The sensitivity of soybean (Glycine max L. Merr. cv Maple Arrow) seeds to dehydration changed during germination. Seeds were tolerant of dehydration to 10% moisture if dried at 6 hours of imbibition, but were susceptible to dehydration injury if dried at 36 hours of imbibition. Dehydration injury appeared as loss of germination, slower growth rates of isolated axes, hypocotyl and root curling, and altered membrane permeability. Increased electrolyte leakage due to dehydration treatment was observed only from isolated axes but not from cotyledons, suggesting that cotyledons are more tolerant of dehydration. The transition from a dehydration-tolerant to a dehydration-susceptible state coincided with radicle elongation. However, the prevention of cell elongation by osmotic treatment in polyethylene glycol (−6 bars) or imbibition in 20 micrograms per milliliter cycloheximide did not prevent the loss of dehydration tolerance suggesting that neither cell elongation nor cytoplasmic protein synthesis was responsible for the change in sensitivity of soybean seeds to dehydration. Furthermore, the rate of dehydration or rate of rehydration did not alter the response to the dehydration stress.     

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl₂. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.     

Cytokinin Regulation of Flower and Pod Set in Soybeans (Glycine max(L.) Merr.)

Exogenous application of cytokinin to raceme tissues of soybean(Glycine max(L.) Merr.) has been shown to stimulate flower productionand to prevent flower abortion. The effects of these hormoneapplications have been ascertained for treated tissues, butthe effects of cytokinins on total seed yields in treated plantshave not been evaluated. Our objectives were to examine theeffects of systemic cytokinin applications on soybean yieldsusing an experimental line of soybeans, SD-87001, that has beenshown to be highly sensitive to exogenous cytokinin application.Soybeans were grown hydroponically or in pots in the greenhouse,and 6-benzylaminopurine (BA) was introduced into the xylem streamthrough a cotton wick for 2 weeks during anthesis. After theplants had matured, the number of pods, seeds per pod, and thetotal seed weight per plant were measured. In the greenhouse,application of 3.4 x 10^-7 moles of BA resulted in a 79% increasein seed yield compared with controls. Results of field trialsshowed much greater variability within treatments, with consistent,but non-significant increases in seed number and total yieldsof about 3%. Data suggest that cytokinin levels play a significantrole in determining total yield in soybeans, and that increasingcytokinin concentrations in certain environments may resultin increased total seed production. Copyright 2001 Annals ofBotany Company Glycine max, soybean, flower abortion, cytokinin, 6-benzylaminopurine, hydroponic, seed yield, wicking     

Cadaverine, an Essential Diamine for the Normal Root Development of Germinating Soybean (Glycine max) Seeds

When the polyamine content of soybean (Glycine max) seeds was examined during the early stages of germination, the major polyamine in the cotyledons was found to be spermidine, followed by spermine; while very low concentrations of cadaverine were found. In the embryonic axes, however, cadaverine was the main polyamine and its content markedly increased 24 hours after the start of germination. When the germination of the seeds was performed in the presence of 1 millimolar α-difluoromethylornithine (DFMO), a marked decrease in the cadaverine content was found, while the other polyamines were not affected. This decrease of the cadaverine content was already noticeable after the first hours of germination. In the presence of DFMO, a pronounced elongation in the roots of the seedlings and a marked decrease in the appearance of secondary roots as compared with controls, was observed. This abnormal rooting of the seedlings caused by DFMO was almost completely reverted by the addition of 1 millimolar cadaverine. The latter also increased the appearance of secondary roots in the seedlings. The decrease in the cadaverine content produced by DFMO could be traced to a strong inhibition of lysine decarboxylase. A temporal correlation between the increase in cadaverine content and the increase in lysine decarboxylase activity was found. Both reached a maximum at the second day of germination. The activity of diamine oxidase, the cadaverine degrading enzyme, started to increase at the third day and reached a maximum between the fourth and fifth day of germination. DFMO increased the activity of diamine oxidase by about 25%. Hence, the large decrease in cadaverine content produced by DFMO has to be attributed to the in vivo suppression of lysine decarboxylase activity. Ornithine decarboxylase activity was also suppressed by DFMO, but putrescine and spermidine contents were not affected, except in the meristematic tissues. The obtained results suggest an important role for cadaverine in the normal rooting process of soybean seedlings.     

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F → E → C → A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.     

Whole Leaf Carbon Exchange Characteristics of Phosphate Deficient Soybeans (Glycine max L.)

Low phosphate nutrition results in increased chlorophyll fluorescence, reduced photosynthetic rate, accumulation of starch and sucrose in leaves, and low crop yields. This study investigated physiological responses of soybean (Glycine max [L.] Merr.) leaves to low inorganic phosphate (Pi) conditions. Responses of photosynthesis to light and CO₂ were examined for leaves of soybean grown at high (0.50 millimolar) or low (0.05 millimolar) Pi. Leaves of low Pi plants exhibited paraheliotropic orientation on bright sunny days rather than the normal diaheliotropic orientation exhibited by leaves of high Pi soybeans. Leaves of plants grown at high Pi had significantly higher light saturation points (1000 versus 630 micromole photons [400-700 nanometers] per square meter per second) and higher apparent quantum efficiency (0.062 versus 0.044 mole CO₂ per mole photons) at ambient (34 pascals) CO₂ than did low Pi leaves, yet stomatal conductances were similar. High Pi leaves also had significantly higher carboxylation efficiency (2.90 versus 0.49 micromole CO₂ per square meter per second per pascal), a lower CO₂ compensation point (6.9 versus 11.9 pascals), and a higher photosynthetic rate at 34 pascals CO₂ (19.5 versus 6.7 micromoles CO₂ per square meter per second) than did low Pi leaves. Soluble protein (0.94 versus 0.73 milligram per square centimeter), ribulose-1,5-bisphosphate carboxylase/oxygenase content (0.33 versus 0.25 milligram per square centimeter), and ribulose-1,5-bisphosphate carboxylase/oxygenase specific activity (25.0 versus 16.7 micromoles per square meter per second) were significantly greater in leaves of plants in the high Pi treatment. The data indicate that Pi stress alters the plant's CO₂ reduction characteristics, which may in turn affect the plant's capacity to accommodate normal radiation loads.     

Canopy and Seasonal Profiles of Nitrate Reductase in Soybeans (Glycine max L. Merr.)

Nitrate reductase activity of soybeans (Glycine max L. Merr.) was evaluated in soil plots and outdoor hydroponic gravel culture systems throughout the growing season. Nitrate reductase profiles within the plant canopy were also established. Mean activity per gram fresh weight per hour of the entire plant canopy was highest in the seedling stage while total activity (activity per gram fresh weight per hour times the total leaf weight) reached a maximum when plants were in the full bloom to midpod fill stage. Nitrate reductase activity per gram fresh weight per hour was highest in the uppermost leaf just prior to full expansion and declined with leaf position lower in the canopy. Total nitrate reductase activity per leaf was also highest in the upper-most fully expanded leaf during early growth stages. Maximum total activity shifted to leaf positions lower in the plant canopy with later growth stages.     

大豆脂肪氧化酶及Kunitz胰蛋白酶抑制剂缺失种质的创新

脂肪氧化酶和胰蛋白酶抑制剂是大豆蛋白中2种重要的抗营养因子。以黄淮海主栽品种鲁豆4号、中品661、豫豆8号、91D15、潍8640作母本,美国引进缺失Kunitz胰蛋白酶抑制剂品种P．I．L83-4387和优良品种Century缺失脂肪氧化酶的近等位基因系Century-2、Century-2．3和Century-1．3作父本进行有性杂交,利用大豆脂肪氧化酶缺失基因及胰蛋白酶抑制剂缺失基因的生化标记对杂种后代进行多年辅助选择,培育脂肪氧化酶缺失基因(lx1、lx2、lx3)、胰蛋白酶抑制剂缺失基因(ti)等优质多基因聚合的大豆新种质,为我国大豆品质育种、生产及加工利用提供优异种质材料。     

Wound-induced Accumulation of Trypsin Inhibitor Activities in Plant Leaves: Survey of Several Plant Genera

Asexual embryogenesis in Daucus carota L. `Queen Anne's Lace' callus was suppressed by Ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid (2,4-D). The Ethephon effect could be attributed to volatile and nonvolatile substances. The volatile component was probably entirely ethylene. Ethylene was liberated in the cultures in direct proportion to Ethephon added to the medium. Autoclaving of Ethephon caused a substantial decrease of measurable ethylene. Continuous exposure of callus to 5 μl/l ethylene depressed somatic cell embryogenesis, but not markedly. Depression of embryogenesis by 2,4-D was unrelated to ethylene evolution.     

Ultrastructural localization of Kunitz inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method

The soybean trypsin inhibitor (SBTI, Kunitz type) was localized by immunofluorescence and, at the ultrastructural level, by the protein A gold method on thin sections of Glycine max (soybean) cv. Maple Arrow. SBTI was localized in cell walls, protein bodies, the cytoplasm between the lipid-containing spherosomes, and the nucleus of the cotyledon and embryonic axis. In the nucleus, SBTI was present in the chromatin deposit and the nucleolus. The intensity of marking by the gold method decreased in the cell wall from the center of the cotyledon to the periphery. In four-days-old seedlings marking intensity of cell wall was much reduced. No SBTI could be detected in the hypocotyl. In two lines lacking soybean agglutinin (Norredo, T-102) the location of SBTI was similar to that observed in Maple Arrow. In P.I. 196168, a line lacking SBTI, marking intensity of the organelles was reduced to a very low level. Although this study was not designed to discern the cellular function of SBTI, if any, it may establish criteria consistent with its role in soybean.