Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations

The I locus controls inhibition of anthocyanin accumulation in the epidermal cells of the soybean seed coat and affects abundance of PRP1, a proline-rich cell wall protein in the seed coat. Saline-soluble PRP1 is abundant in the developing seed coats of cultivar Richland (homozygous I, yellow), while it is significantly decreased in the pigmented isogenic mutant T157 (homozygous i, imperfect black). In this report, we examined soluble PRP1 in several cultivars containing alleles of the I locus which affect spatial distribution of pigmentation in the seed coat. We also characterized PRP1 in isolines with allelic variants of several other loci involved in seed coat pigmentation, including T and Im. The T gene is pleiotropic and affects both pubescence color and seed coat pigmentation and structure. Soluble PRP1 was abundant in the developing seed coats of lines with yellow seed (I or i ⁱ alleles) regardless of pubescence color, just as in Richland. Likewise, soluble PRP1 was decreased in pigmented seed coats (i ^k or i alleles) with grey (t) pubescence, as in T157. However, the total seed coat proteins were not extractable from pigmented seed coats with tawny pubescence (i, T genotypes) because they have proanthocyanidins that exhibit tannin properties. The dominant Im allele inhibits seed coat mottling (irregular patches of pigmentation) that occurs if plants are infected with soybean mosaic virus. PRP1 was 35 kDa in mottled (im) isolines and 34 kDa in non-mottled (Im) isolines. PRP2, which is expressed later in seed coat development and in the hypocotyl hooks of soybean seedlings, was also smaller in Im isolines. In summary, some of the anthocyanin mutations affect the quantity of soluble PRP1 polypeptides, while others correlate with structural changes in developmentally regulated proline-rich proteins.     

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.     

Duplications That Suppress and Deletions That Restore Expression from a Chalcone Synthase Multigene Family

Seed coat color in soybean is determined by four alleles of the classically defined / (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the / locus, we demonstrated that the / locus is a region of chalcone synthase (CHS) gene duplications. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the / locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silencing and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant ii allele (yellow seed coats with pigmented hila) to the recessive i allele (fully pigmented) can be associated with the absence of a 2.3-kb Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutations exhibit deletions in the CHS4 promoter region. The dominant / allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridizes with both the CHS coding region and CHS1 promoter-specific probes. Mutations of the dominant / allele to the recessive i allele (pigmented seed coats) give rise to 10.4- or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHS1 promoter. Finally, gene expression analysis demonstrated that heterozygous plants (I/i) with yellow seed coats have reduced mRNA levels, indicating that the 12.1-kb Hindlll CHS fragment associated with the dominant / allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed in seed coats.     

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.     

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.     

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.

     

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.     

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.     

Genetic regulation of flavonoid content in seeds and seedlings of Melilotus alba

Chemical analysis of seeds and seedlings of the CC and cc genotypes in Melilotus alba indicated that these alleles affect flavonoid biosynthesis. The CC seed coats contained orientin and iso-orientin, which were absent in the cc seed coats. The pigment responsible for the red pigmentation of young seedlings of CC genotypes was a cyanidin glycoside. The embryos of seeds of both the CC and cc genotypes contained a flavonoid tentatively identified as a 6,8-di-C-pentosylapigenin. The observation that 3′,4′-dihydroxyflavonoids were absent in the cc genotype and that 4′-hydroxyflavonoids were present in both genotypes indicated that the C/c alleles controlled the 3′-hydroxylation of flavonoids. The C/c alleles did not, however, control 3′-hydroxylation of cinnamic acids since caffeic acid was detected in both genotypes.     

Nuclear and cytoplasmic "stay-green" mutations of soybean alter the loss of leaf soluble proteins during senescence

In soybean ( Glycine max [L.] Merr.) the homozygous combination of the recessive alleles dI and d2 (i.e., dldld2d2 ) at two different nuclear loci or the cytoplasmic gene cytG inhibit chlorophyll degradation during senescence; i.e. their leaves are green when they are shed. The main objectives of the present work were: (J) to determine whether these "stay-green" genes also interfere with the loss of the bulk of leaf soluble proteins and ribulose bisphospnate carboxylase/oxygensase (Rubisco; EC 4.1.1.39) during senescence and (2) to relate this to alterations in leaf proteolytic activity. Leaves of the normal. Yellowing cvs Clark and Harosoy lost about 90% of their soluble proteins before abscission. The abscising leaves of these cultivars contained no detectable Rubisco. By contrast, protein degradation was significantly less in leaves of near-isogenic lines of Clark and Harosoy carrying dIdId2d2 , with or without G (a dominant nuclear gene in a third locus causing green seed coats). These leaves still retained 50% of the soluble protein and large amounts of both subunits of Rubisco at the time of abscission. Alone, neither dl nor d2 had any effect. The cytoplasmic gene cytG slowed the loss of Rubisco. although eventually when leaves were shed they contained as little Rubisco as Clark. Despite inhibition (i.e. dIdId2d2 and GGdIdId2d2 ) or retardation (i.e. cytG ) of protein loss, these mutant genotypes did not differ from Clark in the breakdown of endogenous Rubisco by leaf extracts ("autodigestion"). The wild-type alleles in the dI and d2 loci may control a central regulatory process of the senescence syndrome.     

Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase

Three loci (I, R, and T) control pigmentation of the seed coats in Glycine max and are genetically distinct from those controlling flower color. The T locus also controls color of the trichome hairs. We report the identification and isolation of a flavonoid 3' hydroxylase gene from G. max (GmF3'H) and the linkage of this gene to the T locus. This GmF3'H gene was highly expressed in early stages of seed coat development and was expressed at very low levels or not at all in other tissues. Evidence that the GmF3'H gene is linked to the T locus came from the occurrence of multiple RFLPs in lines with varying alleles of the T locus, as well as in a population of plants segregating at that locus. GmF3'H genomic and cDNA sequence analysis of color mutant lines with varying t alleles revealed a frameshift mutation in one of the alleles. In another line derived from a mutable genetic stock, the abundance of the mRNAs for GmF3'H was dramatically reduced. Isolation of the GmF3'H gene and its identification as the T locus will enable investigation of the pleiotropic effects of the T locus on cell wall integrity and its involvement in the regulation of the multiple branches of the flavonoid pathway in soybean.     

Sequence divergence at chalcone synthase gene in pigmented seed coat soybean mutants of the Inhibitor locus

Seed coat color in soybeans is determined by the I (Inhibitor) locus. The dominant I allele inhibits seed coat pigmentation, and it has been suggested that there is a correlation between the inhibition of pigmentation by the I allele and chalcone synthase (CHS) gene silencing in the seed coat. Analysis of spontaneous mutations from I to i has shown that these mutations are closely related to the deletion of one of the CHS genes (designated ICHS1). In soybeans with the I/I genotype (cv. Miyagi shirome), a truncated form of the CHS gene (CHS3) is located in an inverse orientation 680 bp upstream of ICHS1, and it was previously suggested that the truncated CHS3- ICHS1 cluster might be involved in CHS gene silencing in the seed coat. In the current study, the truncated CHS3- ICHS1 cluster was compared with the corresponding region of pigmented seed coat mutants in which I had changed to i in Miyagi shirome and in the strain Karikei 584. In the Karikei 584 mutant, the truncated CHS3-ICHS1 cluster was retained and the sequence diverged at a point immediately upstream (32 bp) of this cluster. The sequences upstream of the points of divergence in both mutants almost perfectly matched a part of the registered sequence in a soybean BAC clone containing the soybean cyst nematode resistance-associated gene, and inspection of the sequences suggested that the sequence divergence of the CHS gene in the Karikei 584 and Miyagi shirome mutants was due to an unequal crossing-over via 4-bp or 5-bp short repeats, respectively.     

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.     

An analysis of the distribution of clone cells in the retinal pigment epithelium from chimeric mice

The quantity of pigmented and unpigmented cells was estimated in the retinal pigment epithelium (RPE) in ten newborn and five 20-days old aggregated chimaeric mice C/C----c/c. A strong correlation was shown in the proportion of cells of the paternal genotypes either in the whole RPE of right and left eyes or in its separate regions, i.e. dorsal, central, ventral. Random distribution was revealed in these RPE cells clones. A high correlation was shown between the number of RPE pigmented cells and percentage of coat pigmentation.     

Some morphometric,anatomical and biochemical characteristics of fruits and seeds of Onobrychis spp. in Italy

ABSTRACT

The fruits, seed coats and seed storage proteins of Onobrychis arenaria, O. montana, O. viciifolia, O. alba, O. supina and O. caput-galli were studied to examine the variability between and within species. The morphometric characteristics of the fruit had a high intraspecific variability which often exceeded that between species. Only the fruit of O. caput-galli had values for length, width, thickness and number of thorns which were almost always higher than those of the other species. The anatomical characteristics of the seed coats were extremely variable. The highest values of seed coat thickness were recorded in the diploid species. The palisade-like layer (or Malpighian cells) in O. caput-galli differed in size and morphology from that of the other species. The electrophoretic analysis revealed that the number and position of storage proteins varied between and within species.     

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.