Immobilisation of soybean seed coat peroxidase on its natural support for phenol removal from wastewater

Soybean seed coat peroxidase (SBP; EC 1.11.1.7) was immobilised on its natural support, soybean seed coats, anticipating its use in phenol removal. Periodate and glutaraldehyde chemistries were assayed. Periodate failed to immobilise any SBP, whereas glutaraldehyde was effective. The optimum concentration of glutaraldehyde was found to be 1%. Immobilisation shifted the optimum pH for phenol removal from 4.0 to 6.0. Treated seed coat retained its activity over a 4-week period, and reusability assays showed that treated seed coats could be reused once for phenol removal. Polyethylene glycol (PEG) increased the stability of phenol degradation activity. In addition, the phenolic polymer was adsorbed on to seed coats, thus making removal of the polymeric product easier.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP?g^?1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^?1 µg^?1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Extraction of RNA from tissues containing high levels of procyanidins that bind RNA

Commonly used methods for extraction of RNA from plants are not effective for isolation of high quality RNA from the pigmented seed coats of soybeans that produce procyanidins (tannins) during seed coat development. We demonstrate a significant modification of the phenol-LiCl method that yields high quality RNA from a black seed coat variety. In this method, seed coat material was ground in a buffer containing a high concentration of bovine serum albumin (100 mg BSA/50 mg of lyophilized seed coats) to competitively inhibit proanthocyanidin binding. The presence of hydrated insoluble polyvinylpoly-pyrrolidone (PVPP) was also necessary to bind proanthocyanidins and remove them from solution. Proteinase K was added to digest the remaining BSA, and phenol extraction was used to remove both the proteins and small molecular weight complexes formed by BSA and proanthocyanidins. After LiCl and ethanol precipitations, the RNA quality was examined by UV absorbance spectra, gel electrophoresis, and hybridization. Using this method, good quality RNA can be extracted from pigmented seed coats of soybean varieties that are homozygous for the recessivei allele and also contain the dominantT gene that results in production of procyanidins in the seed coat. The method is also effective for tissues from other plant species that contain abundant polyphenolic compounds.     

Soybean Seed Coat Peroxidase (A Comparison of High-Activity and Low-Activity Genotypes)

Peroxidase activity in the seed coats of soybean (Glycine max [L.] Merr.) is controlled by the Ep locus. We compared peroxidase activity in cell-free extracts from seed coat, root, and leaf tissues of three EpEp cultivars (Harosoy 63, Harovinton, and Coles) to three epep cultivars (Steele, Marathon, and Raiden). Extracts from the seed coats of EpEp cultivars were 100-fold higher in specific activity than those from epep cultivars, but there was no difference in specific activity in crude root or leaf extracts. Isoelectric focusing of root tissue extracts and staining for peroxidase activity showed that EpEp cultivars had a root peroxidase of identical isoelectric point to the seed coat peroxidase, whereas roots of the epep types were lacking that peroxidase, indicating that the Ep locus may also affect expression in the root. In seed coat extracts, peroxidase was the most abundant soluble protein in EpEp cultivars, whereas this enzyme was present only in trace amounts in epep genotypes, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Histochemical localization of peroxidase activity in seed coats of EpEp cultivars shows that the enzyme occurs predominately in the cytoplasm of hourglass cells of the subepidermis. No obvious difference in the gross or microscopic structure of the seed coat was observed to be associated with the Ep locus. These results suggest that soybean seed coat peroxidase may be involved in processes other than seed coat biosynthesis.     

Changes in phenolic acids and abscisic acid in sugar pine seed coats during stratification

The phenolic acids and abscisic acid (ABA) of sugar pine ( Pinus lambertiana Dougl.) seeds coats, separated by high-pressure liquid chromatography, were analyzed during 90 days stratification of the seeds. Although levels of seed coat phenolic acids and ABA declined significantly during, stratification, this decrease did not appear to be responsible for the loss of dormancy due to stratification. Lack of improved germination following washing, cracking, or removal of the seed coats, plus additional evidence, did not support a significant role for the seed coat in the dormancy of sugar pine seeds.     

Soybean peroxidase propeptides are functional signal peptides and increase the yield of a foreign protein

Elements that contribute to the high, stable yield of soybean peroxidase (SBP) in soybean seed coats can be exploited in the development of this tissue as a protein production platform. SBP contains an N-terminal and a C-terminal propeptide that are predicted to direct vacuolar targeting; this may be one factor that contributes to its high yield and stability. We characterized the function of the SBP propeptides and investigated their ability to increase the yield of a foreign protein in a heterologous plant system. SBP propeptides are functional signal peptides capable of directing vacuolar transport in Arabidopsis. The use of these propeptides as well as an endoplasmic reticulum (ER)-retention signal to direct a foreign protein to the apoplast, ER, or vacuole can significantly increase yield and will therefore be useful for the development of the seed coat as a protein production platform. We also demonstrate that growth conditions may have a significant impact on the yield of a foreign protein and that this may be subcellular compartment-specific.     

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.     

Changes in Photosynthate Unloading from Perfused Seed Coats of Phaseolus vulgaris L. Induced by Osmoticum and Ethylenediaminetetraacetate (EDTA)

Photosynthate unloading in Phaseolus vulgaris L. seed coatswas studied by treating perfused seed coats with differing concentrationsof an osmoticum and ethylenediaminetetraacetate (EDTA). Largechanges in osmoticum concentration typically produced rapidchanges in efflux of unlabelled sugar and steady-state-labelled¹⁴C-photosynthate. Osmoticum-induced changes in photosynthateefflux were caused by phloem import stimulation at low cellturgor and net efflux stimulation by high cell turgor. Eventhough rapid changes in sugar and tracer efflux were often inducedby osmoticum treatments, the specific activity of sugar releasedfrom seed coats was not greatly affected by these treatmentsand was similar to the specific activity of sugar remainingin the seed coat after perfusion. Thus, tracer was transportedfrom the phloem throughout the seed coat sugar pool before itwas released to the apoplast. This result is most consistentwith symplastic phloem unloading throughout perfused seed coats,because apoplastic transport between cells within the seed coatwas blocked by perfusion. Photosynthate efflux was stimulatedby simultaneous treatment of seed coats with EDTA and differentconcentrations of an osmoticum; loss of photosynthate from seedcoats did not appear to be tissue-specific. Key words: Phaseolus vulgaris, seed coat, photosynthate unloading, turgor, EDTA     

Seed coat composition in black and white soybean seeds with differential water permeability

• The seed coat composition of white (JS 335) and black (Bhatt) soybean (Glycine max (L.) Merr) having different water permeability was studied.
• Phenols, tannins and proteins were measured, as well as trace elements and metabolites in the seed coats.
• The seed coat of Bhatt was impermeable and imposed dormancy, while that of JS 335 was permeable and seeds exhibited imbibitional injury. Bhatt seed coats contained comparatively higher concentrations of phenols, tannins, proteins, Fe and Cu than those of JS 335. Metabolites of seed coats of both genotypes contained 164 compounds, among which only 14 were common to both cultivars, while the remaining 79 and 71 compounds were unique to JS 331 and Bhatt, respectively.
• Phenols are the main compounds responsible for seed coat impermeability and accumulate in palisade cells of Bhatt, providing impermeability and strength to the seed coat. JS 335 had more cracked seed coats, mainly due to their lower tannin content. Alkanes, esters, carboxylic acids and alcohols were common to both genotypes, while cyclic thiocarbamate (1.07%), monoterpene alcohols (1.07%), nitric esters (1.07%), phenoxazine (1.07%) and sulphoxide (1.07%) compounds were unique to the JS 335 seed coat, while aldehydes (2.35%), amides (1.17%), azoles (1.17%) and sugar moieties (1.17%) were unique to Bhatt seed coats. This study provides a platform for isolation and understanding of each identified compound for its function in seed coat permeability.

     

Peroxidase involvement in lignification in water-impermeable seed coats of weedy leguminous and malvaceous species

Abstract. The development of water impermeable seed coats of two members each of the leguminoseae family [ Crotalaria spectabilis Roth, Sesbania exaltata (Raf) Cory] and the malvaceae family [Anoda cristata (L.) Schlecht, Abutilon theophrasti Medic.] was investigated. Highest peroxidase (POD) activity of Anoda and Abutilon seed coat extracts was highly correlated with the developmental stages when soluble phenolics were maximally converted into lignin. Although extensive lignification occurred during seed coat development in both legumes, the patterns of POD activity, soluble phenolic levels and time of lignification were different from those of the malvaceous species. POD activity levels in developing coats of the malvaceous seeds increased as phenolics decreased. Both POD activity and phenolic levels decreased during seed coat development of the legumes. POD was immunocytochemically and immunochemically detected in seed coats of all four species; however, results for polyphenol oxidase were negative. The results confirmed POD involvement in lignification of leguminous and malvaecous species and support and extend our earlier view that POD is involved in lignin formation during development of impermeable seed coats.     

Enzymatic Removal of Phenol and Chlorophenols Using Soybean Seed Hulls

Soybean peroxidase (SBP), (EC 1.11.1.7) can be readily extracted from soybean seed hulls. This study reports on the direct use of soybean seed‐hull extracts for the bioremediation of phenolic wastes. The crude SBP extract from the hulls, like pure soybean peroxidase, is catalytically active in a broad range of pH and temperatures. As SBP is gradually released into the aqueous solution from seed hulls, the direct use of soybean seed hulls can reduce SBP inactivation by H₂O₂ and enhance the utilization efficiency of SBP through the slow release of the enzyme from the seed hulls. However, large doses of soybean seed hulls were found to be ineffective in phenol removal. Gradual additions of H₂O₂ in combination with the SBP released from the hulls were applied to optimize the bioremediation. Since the crude extract contains a mixture of multiple soybean proteins, soybean seed hull slurry required a higher concentration of H₂O₂ to remove the phenolic substrates than did the purified enzyme. Under the experimental conditions, 80 % of phenol (10.6 mM), 96 % of 2‐chlorophenol (3.9 mM), 95 % of 2,4‐dichlorophenol (3.1 mM), and 94 % of mixed phenol and chlorophenols were removed using soybean seed hulls in a single batch reactor. These results demonstrate that soybean seed hulls, compared to purified SBP, may be a more cost‐effective alternative in the enzymatic removal of phenolic compounds through polymerization reactions.     

Patterns and kinetics of water uptake by soybean seeds

Soybean [Glycine max (L.) Merr.] plants produce some seeds (called stone or impermeable seeds) that do not take up water for long periods of time. The present investigation confirmed that the stone seed trait is a feature of the seed coat: isolated embryos from both stone and permeable seeds took up water equally quickly. A whole, permeable seed typically imbibed water initially through its dorsal side, forming wrinkles in the seed coat and delivering water to the underlying cotyledons. Later, some lateral movement of water through the coat occurred, presumably through the air spaces of the osteosclereid layer. Imbibition by seeds was a two-phase process, the first dominated by hydration of the seed coat and the second by hydration of the cotyledons, which was rate-limited by the coat. When hydrated, coats of stone seeds were permeable to water but their hydraulic conductivity, as measured with a pressure probe, was smaller than that of coats from permeable seeds by a factor of five. Hydrated coats of both permeable and stone seeds showed weak osmometer properties.     

Quantitative Analysis of Photosynthate Unloading in Developing Seeds of Phaseolus vulgaris L. : II. Pathway and Turgor Sensitivity

Phloem import and unloading in perfused bean (Phaseolus vulgaris L.) seed coats were investigated using steady-state labeling. Though photosynthate import and unloading were significantly reduced by perfusion, measurements of photosynthate fluxes in perfused seed coats proved useful for the study of unloading mechanisms in vivo. Phloem import was stimulated by lowered seed coat cell turgor, as demonstrated by an increase in tracer and sucrose import to seed coats perfused with high concentrations of an osmoticum. The partitioning of photosynthates between retention in the seed coat and release to the perfusion solution also was turgor sensitive; increases in seed coat cell turgor stimulated photosynthate release to the apoplast at the expense of photosynthate retention within the seed coat. There was no evidence of a turgor-sensitive sucrose uptake mechanism in perfused seed coats. Thus, the turgor sensitivity of photosynthate partitioning within perfused seed coats was consistent with a turgor-sensitive efflux control mechanism. Measurements of tracer equilibration and sugar partitioning in perfused seed coats provided strong evidence for symplastic phloem unloading in seed coats.     

Differences in release of endogenous sugars and amino acids from attached and detached seed coats of developing pea seeds

The effect of p -chloromercuribenzenesulfonic acid (PCMBS), carbonylcyanide- m -chlorophenylhydrazone (CCCP) and a high apoplastic pH (pH 7.5 compared with pH 5.5) on the release of sugars (sucrose and glucose) and amino acids from attached and detached seed coats of Pisum sativum L. cv. Marzia into a bathing solution was measured by means of the 'empty seed coat technique'. PCMBS reduced the release of sugars and amino acids from attached as well as from detached seed coats, suggesting that carrier-mediated transport might be involved. CCCP reduced sugar release from attached seed coats while amino acid release was hardly affected. In experiments with detached seed coats CCCP had no effect on release of either sugar or amino acids, suggesting that it is not energy-dependent. Raising the pH of the bathing solution from pH 5.5 to pH 7.5 slightly increased sugar release from both attached and detached seed coats while amino acid release was not affected. This might indicate a role of the apoplastic pH in regulating sugar release from the seed coat via a retrieval mechanism. The presented data indicate that there are important differences between sugars and amino acids with respect to transport processes in the seed coat. This is supported by the observation that the rate of amino acid release from the seed coat was higher than the rate of sugar release. The release data of detached seed coats were subjected to compartmental analysis in order to calculate rate constants for release from cell compartments. In the case of sugars, the half-times for emptying the cytoplasmic and vacuolar compartment were 0.8 h and 12.5 h. respectively. For amino acids the half-times were 0.5 h for emptying the cytoplasmic and 3.8 h for emptying the vacuolar compartment.     

Osmotic regulation of assimilate unloading from seed coats of Vicia faba. Assimilate partitioning to and within attached seed coats.

The significance of the osmotic potential of the seed apoplast sap as a regulator of assimilate transfer to and within coats of developing seed of Vicia faba (cv. Coles Prolific) was assessed using attached empty seed coats and intact developing seed. Following surgical removal of the embryos, through windows cut in the pod walls and underlying seed coats, the resulting attached “empty” seed coats were filled with solutions of known osmotic potentials (–0. 02 versus –0. 75 MPa). Sucrose efflux from the coats was elevated at the higher osmotic potential (high osmotic concentration) for the first 190 min of exchange. Thereafter, this efflux was depressed relative to efflux from coats exposed to the low osmotic potential (high osmotic concentration) solution. This subsequent reversal in efflux was attributable to an enhanced diminution of the coat sucrose pools at the high external osmotic potential. Indeed, when expressed as a proportion of the current sucrose pool size, relative efflux remained elevated for coats exposed to the high osmotic potential solution. Measurement of potassium and sucrose fluxes to and from their respective pools in the coat tissues demonstrated that the principal, fluxes, sensitive to variative in the external osmotic potential, were phloem import into and efflux from the “empty” coats. Phloem import, consistent with a pressure-driven phloem transport mechanism, responded inversely with changes in the external osmotic potential. In contrast, sucrose and potassium efflux from the coats exhibited a positive dependence on the osmotic potential. Growth rates of whole seed were approximately doubled by enclosing selected pods in water jackets held at temperatures of 25°C. compared to 15°C. The osmotic potential of sap collected from the seed apoplast remained constant and independent of the temperature-induced changes in seed growth rates and hence phloem import. Based on these findings, it is proposed that control of phloem import by changes in the external osmotic potential observed with “empty” seed coats has no significance as a regulator of assimilate import by intact seed. Rather, maintenance of the seed apoplast osmotic potential, independent of seed growth rate, suggests that the observed osmotic regulation of efflux from the coats may play a key role in integrating assimilate demand by the embryo with phloem import.     

掌叶木种皮障碍及种子各部位内源抑制活性的研究

掌叶木(Handeliodendron bodinieri)是残遗于中国的稀有单种属植物,因人为破坏、生境特殊及自身特性的影响,资源稀少,被列为国家一级重点保护野生珍稀濒危植物。该研究以掌叶木种子为材料,研究了4种不同发芽条件下(带种皮、浓硫酸处理种皮、完全去除种皮、仅露出胚根)种子萌发性、种皮透水性、掌叶木果皮、假种皮、种皮和种仁四个部位不同浓度甲醇浸提物(0、3.125、6.25、12.5、25 mg/m L)对白菜种子萌发及幼苗生长的影响以及掌叶木各部位浸提物对种子萌发的影响。结果表明:(1)掌叶木种皮具有一定的透水性,为掌叶木种子的萌发提供必要的透水透气条件,不影响种子萌发前的水分吸收,但掌叶木种皮的机械阻碍、易霉变对种子的萌发影响较大。(2)掌叶木的果皮、假种皮、种皮和种仁甲醇浸提物对白菜种子的萌发和生长都有影响,尤其对白菜幼根的生长有较强的抑制作用,抑制强度依次是种仁果皮假种皮种皮,且随着浓度的升高,抑制作用增强。该研究结果揭示了掌叶木种子难发芽、发芽率低的原因,为掌叶木的人工扩繁和保护与利用奠定了基础。     

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a "seed coat bedding" assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.     

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.     

Development of seed coat-imposed dormancy during seed maturation in Cynoglossum officinale

The relationship between seed phenolics and appearance of seed coat–imposed dormancy during seed development in Cynoglossum officinale L. was studied. Up to 24 days after anthesis, seeds failed to germinate upon imbibition in Petri dishes at 25°C. At 44 days after anthesis, seeds were fully germinable; removal of seed coats did not improve their germination or O₂ uptake. At 72 days after anthesis, mature seeds at the base of the cyme did not germinate unless their coats were removed. Removal of seed coat also stimulated O₂ uptake at this harvest date. The methanol-soluble phenolic content of the seeds increased during the early stages of seed development, in both the seed coat and the embryo. As seed development continued, the methanol-soluble phenolic content of the embryo stabilized, but that of the seed coat declined. This decline was associated with an increase in the thioglycolic acid–soluble phenolics, presumably lignins, in the seed coat. These results suggest that polymerization of methanol–soluble phenolics into lignins in the seed coat during later stages of seed development renders the seed coat of C. officinale impermeable to 03, and thus keeps the seed dormant.