Two subtilisin-like proteases from soybean

Two subtilisin-like proteases (SLP) were identified in soybean ( Glycine max [L.] Merr.). The first, SLP-1, was localized in seed coats early in seed development, but became undetectable with anti-SLP-1 antibodies as seed fill progressed. A partial purification of SLP-1 was achieved using a two step chromatographic procedure. NH₂-terminal sequence analysis of the partially purified enzyme permitted primers to be designed that were used to amplify cDNA encoding SLP-1. A genomic clone encoding SLP-1 was also obtained. Characterization of the cDNA and partially purified SLP-1 revealed the initial translation product was an 82 694 MW precursor. After removal of a signal peptide, the mature protein was formed by removal of an NH₂-terminal propeptide. A COOH-terminal peptide also appeared to be removed from some of the protease molecules. DNA blot analysis suggested that at least one additional SLP gene was present in soybean. The second gene, SLP-2, was subsequently cloned and characterized. Although the coding regions for SLP-1 and SLP-2 were homologous, their promoters were quite divergent. RT-PCR revealed that SLP-2 message was found in the mature plant and in cotyledons of germinating seeds. Although SLP-2 mRNA could be identified in developing seeds, the message was at least an order of magnitude less abundant than that for SLP-1, and it was mis-spliced such that a chain termination event would preclude obtaining a product. As with SLPs from other organisms, the functions of the soybean proteases are unknown. However, SLP-1 is one of only a few proteins from soybean seed coats that have been described.     

Isolation of tryptophol as an apoptosis-inducing component of vinegar produced from boiled extract of black soybean in human monoblastic leukemia U937 cells

We isolated a novel apoptosis-inducing component, tryptophol, from vinegar produced from boiled extract of black soybean (black soybean vinegar). Compound-6 purified from an ethyl acetate extract of black soybean vinegar using high performance liquid chromatography (HPLC) induced fragmentation of DNA and the development of apoptotic bodies (characteristic physiological features of apoptosis) in U937 cells. By analysis of chemical structure, this active compound was identified as tryptophol. Tryptophol induced apoptosis involving caspase-8 and -3 activation, followed by cleavage of poly (ADP-ribose) polymerase (PARP), as shown by measurement of enzyme activity and immunoblot analysis. The cell viability of normal lymphocytes separated from human blood was less affected by tryptophol, and fragmentation of DNA was not induced in normal lymphocytes. These results indicate that tryptophol isolated from black soybean vinegar inhibited the proliferation of U937 cells by inducing apoptosis via a pathway involving caspase-8 followed by caspase-3, without affecting normal lymphocytes.     

Purification of allantoinase from soybean seeds and production and characterization of anti-allantoinase antibodies.

Allantoinase catalyzes the hydrolysis of allantoin to allantoic acid, a reaction important in both biogenesis and degradation of ureides. Ureide production in cotyledons of germinating soybean (Glycine max L.) seeds has not been studied extensively but may be important in mobilizing nitrogen reserves. Allantoinase was purified approximately 2500-fold from a crude extract of soybean seeds by differential centrifugation, heat treatment, ammonium sulfate fractionation, ethanol fractionation, and fast protein liquid chromatography (Pharmacia) with Mono-Q and Superose columns. The purified enzyme had a subunit size of 30 kD. Polyclonal antibodies produced against the purified protein titrated allantoinase activity in a crude extract of seed proteins. Antibodies recognized the 30-kD band in western blot analysis of crude seed extracts, indicating that they were specific for allantoinase.     

Allene Oxide Synthase and Allene Oxide Cyclase,Enzymes of the Jasmonic Acid Pathway,Localized in Glycine max Tissues

Because jasmonic acid regulates a number of processes, including the expression of vegetative storage proteins in soybean (Glycine max L.) leaves, the relative activity of a specific portion of the jasmonic acid biosynthetic pathway in soybean tissues was examined. Allene oxide synthase and allene oxide cyclase were examined because they constitute a branch point leading specifically from 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid to 12-oxo-phytodienoic acid, the precursor of jasmonic acid. From growing plants, seed coats (hila plus testae) of green fruits (38 d post-anthesis) were most active, eliciting about 1.5 times greater activity on a per milligram of protein basis than the next most active tissue, which was the pericarp. Leaves from fruiting plants were only one-seventh as active as seed coats, and activities in both immature cotyledons and embryonic axes were very low. No activity was detected in any part of stored, mature seeds. After 72 h of germination of stored seeds, a small amount of activity, about 4% of that in immature seed coats, was found in the plumule-hypocotyl-root, and no activity was detected in the cotyledons. The high levels of jasmonic acid biosynthetic enzymes in soybean pericarp and seed coat suggest a role for jasmonic acid in the transfer of assimilate to seeds.     

Pigmented Soybean (Glycine max) Seed Coats Accumulate Proanthocyanidins during Development

The dominant I gene inhibits accumulation of anthocyanin pigments in the epidermal layer of soybean (Glycine max) seed coats. Seed-coat color is also influenced by the R locus and by the pubescence color alleles (T, tawny; t, gray). Protein and RNA from cultivars with black (i,R,T) and brown (i,r,T) seed coats are difficult to extract. To determine the nature of the interfering plant products, we examined seed-coat extracts from Clark isogenic lines for flavonoids, anthocyanins, and possible proanthocyanidins by thin-layer chromatography. We show that yellow seed-coat varieties (I) do not accumulate anthocyanins (anthocyanidin glycosides) or proanthocyanidins (polymeric anthocyanidins). Mature, black (i,R,T) and imperfect-black (i,R,t) seed coats contained anthocyanins, whereas mature, brown (i,r,T) and buff (i,r,t) seed coats did not contain anthocyanins. In contrast, all colored (i) genotypes tested positive for the presence of proanthocyanidins by butanol/ HCl and 0.5% vanillin assays. Immature, black (i,R,T) and brown (i,r,T) seed coats contained significant amounts of procyanidin, a 3[prime],4[prime]-hydroxylated proanthocyanidin. Immature, black (i,R,T) or brown (i,r,T) seed-coat extracts also tested positive for the ability to precipitate proteins in a radial diffusion assay and to bind RNA in vitro. Imperfect-black (i,R,t) or buff (i,r,t) seed coats contained lesser amounts of propelargonidin, a 4[prime]-hydroxylated proanthocyanidin. Seed-coat extracts from these genotypes did not have the ability to precipitate protein or bind to RNA. In summary, the dominant I gene controls inhibition of not only anthocyanins but also proanthocyanidins in soybean seed coats. In homozygous recessive i genotypes, the T-t gene pair determines the types of proanthocyanidins present, which is consistent with the hypothesis that the T locus encodes a microsomal 3[prime]-flavonoid hydroxylase.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Abscisic Acid and its relationship to seed filling in soybeans

The effect of exogenous abscisic acid (ABA) on the rate of sucrose uptake by soybean (Glycine max L. Merr.) embryos was evaluated in an in vitro system. In addition, the concentrations of endogenous ABA in seeds of three soybean Plant Introduction (PI) lines, differing in seed size, were commpared to their seed growth rates. ABA (10⁻⁷ molar) stimulated in vitro sucrose uptake in soybean (cv `Clay') embryos removed from plants grown in a controlled environment chamber, but not in embryos removed from field-grown plants of the three PI lines. However, the concentration of ABA in seeds of the three field-grown PI lines correlated well with their in situ seed growth rates and in vitro [¹⁴C] sucrose uptake rates.

Across genotypes, the concentration of ABA in seeds peaked at 8.5 micrograms per gram fresh weight, corresponding to the time of most rapid seed growth rate, and declined to 1.2 micrograms per gram at physiological maturity. Seeds of the large-seeded genotype maintained an ABA concentration at least 50% greater than that of the small-seeded genotype throughout the latter half of seed filling. A higher concentration of ABA was found in seed coats and cotyledons than in embryonic axes. Seed coats of the large-seeded genotype always had a higher concentration of ABA than seed coats of the small-seeded line. It is suggested that this higher concentration of ABA in seed coats of the large-seeded genotype stimulates sucrose unloading into the seed coat apoplast and that ABA in cotyledons may enhance sucrose uptake by the cotyledons.

     

Antioxidant capacity of a 3-deoxyanthocyanidin from soybean.

Soybean cotyledons directly exposed to UV-C (190-280 nm) contained a colored pigment in those areas of the epidermis directly exposed to UV-C. Ethanolic extracts from UV-C irradiated cotyledons showed a significant peak at 532 nm at pH=10, but not seen at pH=6, successive changes in pH were accompanied by reversible changes in the spectra. The identity of the pigment isolated from soybean cotyledons was established as apigeninidin by comparing the features of standard of a apigeninidin (from sorghum) previously characterized by FAB-MS, UV, HPLC, 1H NMR, and IR spectroscopy. To characterize antioxidant activity of this compound, its ability to scavenge radical species in vitro was tested. In the concentration range tested (up to 200 microg ml (-1)), apigeninidin did not show any scavenger activity towards hydroxyl radical, quinones or NO. However, ascorbyl radical and lipid radicals were effectively quenched in a dose-dependent manner. Overall, UV-C radiation triggers molecular signals that lead in soybean cotyledons to the synthesis and accumulation of an antioxidant pigment, apigeninidin, that shows scavenger activity against ascorbyl and lipid radicals in in vitro studies.     

Proton extrusion in seed coats of Phaseolus vulgaris L.

Abstract. The present investigations were designed to identify proton pumps in seed coats of Phaseolus vulgaris L. Vacated seed-coat halves were exposed to bathing solutions with indicators for proton pump action and the pH changes in the media were measured. Fusicoccin increased the rate of proton extrusion from the seed coats. Orthovanadate and abscisic acid retarded the proton extrusion evoked by fusicoccin. Abolition of the proton extrusion by parachloromercuriphenylsulphonic acid was partially reversed by diethioerythritol. The extrusion was stimulated by high osmolarities (100 mol m⁻³ sorbitol), potassium ions (100 mol m⁻³ KCI) and light. Old seed coats reacted more rapidly to fusicoccin treatments than young ones. Proton pumping in seed coats and cotyledons showed differential responses to fusicoccin, K⁺ and sucrose. In contrast to seed coats, medium acidification by cotyledons was prohibited by addition of sucrose. The significance of proton pumps for photosynthate transfer in vivo is discussed.     

Sucrose Concentration at the Apoplastic Interface between Seed Coat and Cotyledons of Developing Soybean Seeds

The apoplastic sucrose concentration at the interface between cotyledons and surrounding seed coats of developing soybeans (Glycine max L. Merr. cv Wye) was found by three indirect methods to be in the range of 150 to 200 millimolar. This is an order of magnitude higher than has been reported elsewhere for soybean. It was also higher than the overall sucrose concentrations in the cotyledons and seed coats, each of which was approximately 90 millimolar. By defoliating plants 24 hours before measurement, both the overall sucrose concentration in the cotyledons and the interfacial apoplastic sucrose concentration were reduced by three-fourths. However, there was no day/night difference in overall tissue sucrose concentration of cotyledons or seed coats from intact plants suggesting the existence of a homeostatic mechanism compensating for the diurnal photosynthetic cycle. About 7 hours were required for a tritiated polyethylene glycol-900 solution to fully permeate developing cotyledons (from ~220 milligram fresh weight embryos), implying high diffusion resistance through the tissue.

These results indicate that a high interfacial sucrose concentration may exist in vivo. They suggest that the saturable carrier-mediated component of sucrose uptake may be of little physiological significance in the outermost cell layers of the cotyledons.

     

An abundant, highly conserved tonoplast protein in seeds

We have isolated the membranes of the protein storage vacuoles (protein bodies) from Phaseolus vulgaris cotyledons and purified an integral membrane protein with M_r 25,000 (TP 25). Antiserum to TP 25 recognizes an abundant polypeptide in the total cell extracts of many different seeds (monocots, dicots, and a gymnosperm), and specifically labels the vacuolar membranes of thin-sectioned soybean embryonic axes and cotyledons. TP 25 was not found in the starchy endosperm of barley and wheat or the seed coats of bean but was present in all seed parts examined that consist of living cells at seed maturity. The abundance of TP 25 was not correlated with the amount of storage protein in seed tissue, and the protein was not found in leaves that accumulate leaf storage protein. On the basis of its abundance, evolutionary conservation, and distribution in the plant, we propose that TP 25 may play a role in maintaining the integrity of the tonoplast during the dehydration/rehydration sequence of seeds.     

A soybean cell wall protein is affected by seed color genotype.

The dominant I gene inhibits accumulation of anthocyanin pigments in epidermal cells of the soybean seed coat. We compared saline-soluble proteins extracted from developing seed coats and identified a 35-kilodalton protein that was abundant in Richland (genotype I/I, yellow) and much reduced in an isogenic mutant line T157 (genotype i/i, imperfect black seed coats). We purified the 35-kilodalton protein by a novel procedure using chromatography on insoluble polyvinylpolypyrrolidone. The 35-kilodalton protein was composed primarily of proline, hydroxyproline, valine, tyrosine, and lysine. Three criteria (N-terminal amino acid sequence, amino acid composition, and sequence of a cDNA) proved that the seed coat 35-kilodalton protein was PRP1, a member of a proline-rich gene family expressed in hypocotyls and other soybean tissues. The levels of soluble PRP1 polypeptides and PRP1 mRNA were reduced in young seed coats with the recessive i/i genotype. These data demonstrated an unexpected and novel correlation between an anthocyanin gene and the quantitative levels of a specific, developmentally regulated cell wall protein. In contrast, PRP2, a closely related cell wall protein, was synthesized later in seed coat development and was not affected by the genotype of the I locus.     

Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominant I alleles.

The seed of all wild Glycine accessions have black or brown pigments because of the homozygous recessive i allele in combination with alleles at the R and T loci. In contrast, nearly all commercial soybean (Glycine max) varieties are yellow due to the presence of a dominant allele of the I locus (either I or i) that inhibits pigmentation in the seed coats. Spontaneous mutations to the recessive i allele occur in these varieties and result in pigmented seed coats. We have isolated a clone for a soybean dihydroflavonol reductase (DFR) gene using polymerase chain reaction. We examined expression of DFR and two other genes of the flavonoid pathway during soybean seed coat development in a series of near-isogenic isolines that vary in pigmentation as specified by combinations of alleles of the I, R, and T loci. The expression of phenylalanine ammonia-lyase and DFR mRNAs was similar in all of the gene combinations at each stage of seed coat development. In contrast, chalcone synthase (CHS) mRNA was barely detectable at all stages of development in seed coats that carry the dominant I allele that results in yellow seed coats. CHS activity in yellow seed coats (I) was also 7- to 10-fold less than in the pigmented seed coats that have the homozygous recessive i allele. It appears that the dominant I allele results in reduction of CHS mRNA, leading to reduction of CHS activity as the basis for inhibition of anthocyanin and proanthocyanin synthesis in soybean seed coats. A further connection between CHS and the I locus is indicated by the occurrence of multiple restriction site polymorphisms in genomic DNA blots of the CHS gene family in near-isogenic lines containing alleles of the I locus.     

Effect of Exogenous Hormones on Endogenous ABA Level with Its Relation to Assimilate Accumulation in Soybean Seeds

Injecting IAA, GA3, and KT into soybean ( Gtycine max L. ) seeds in vivo on intact plant of 30 d after anthesis, the ABA content, invertase and ATPase activity in seed coat and cotyledons were tested respectively. It was revealed that the content of endogenous ABA was correlated with the invertase and ATPase activity. The ABA level increased was 24% and 65 % in seed coat by using 10-6 mol/L LAA and 10-6 moL/L KT and decreased 19% and 41% in cotyledons, respectively. Correlatively, ABA stimulated invert&se activity and inhibited ATPase activity in seed coat, but the reverse effect of ABA was seen in cotyledons. Treated with 10-6 mol/L GA3, ABA level droped by 42% in seed coat and by 22% in cotyledons. Activities of invertase and ATPase were inhibited in seed coat but stimulated in cotyledons. In studying the changes of sugar and protein in the cotyledons, it was shown that ABA was involved in the assimilate accumulation in soybean seeds. The effects of exogenous hormones on endogenous ABA level and ABA in relation to assimilate accumulation in the cotyledons were discussed.     

Demonstration of Aflatoxin Inhibitory Activity in a Cotton Seed Coat Xylan

An inhibitor of aflatoxin biosynthesis localized in the seed coats of developing cotton was partially purified and characterized. Aqueous extracts from 25-day postanthesis seed coat tissue inhibited aflatoxin (B(inf1)) production in liquid cultures of Aspergillus flavus AF13. Inhibition was concentration dependent, with a 50% effective dose of 173 (mu)g of crude extract per ml of medium. The inhibitor was neutral in charge. Two active fractions were obtained from crude preparations by gel filtration chromatography (BioGel P-100). The purest fraction eluted in the void volume. Carbohydrate composition analysis of this void volume inhibitor indicated a composition of xylose (>90%) and mannose. Aflatoxin production in vitro was inversely related to inhibitor concentration in the fermentation medium (log of aflatoxin versus log of [inhibitor]; r(sup2) = 0.82; P < 0.002). The void volume inhibitor had a 50% effective dose of 6.2 (mu)g/ml, a 28-fold purification of the inhibitor material. These data support the hypothesis that seed coat inhibitory activity is associated with a cottonseed-specific xylan.     

A defective seed coat pattern (Net) is correlated with the post-transcriptional abundance of soluble proline-rich cell wall proteins

The pigmented seed coats of several soybean (Glycine max (L.) Merr.) plant introductions and isolines have unusual defects that result in cracking of the mature seed coat exposing the endosperm and cotyledons. It has previously been shown that the T (tawny) locus that controls the color of trichomes on stems and leaves also has an effect on both the structure and pigmentation of the seed coat. Distribution of pigmentation on the seed coat is controlled by alleles of the I (inhibitor) locus. It was also found that total seed coat proteins were difficult to extract from pigmented seed coats with i T genotypes because they have procyanidins that exhibit tannin properties. We report that the inclusion of poly-L-proline in the extraction buffer out-competes proteins for binding to procyanidins. Once this problem was solved, we examined expression of the proline-rich cell wall proteins PRP1 and PRP2 in pigmented genotypes with the dominant T allele. We found that both homozygous i T and i t genotypes have reduced soluble PRP1 levels. The epistatic interaction of the double recessive genotype at both loci is necessary to produce the pigmented, defective seed coat phenotype characteristic of seed coats with the double recessive i and t alleles. This implies a novel effect of an enzyme in the flavonoid pathway on seed coat structure in addition to its effect on flavonoids, anthocyanidins, and proanthocyanidins. No soluble PRP1 polypeptides were detectable in pigmented seed coats (i T genotypes) of isolines that also display a net-like pattern of seed coat cracking, known as the Net defect. PRP2 was also absent in one of the these lines. However, both PRP1 and PRP2 cytoplasmic mRNAs were found in the Net-defective seed coats. Together with in vitro translation studies, these results suggest that the absence of soluble PRP polypeptides in the defective Net lines is post-translational and could be due to a more rapid or premature insolubilization of PRP polypeptides within the cell wall matrix.