A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

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.     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

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.     

Factors Affecting Attachment of Enterobacter cloacae to Germinating Cotton Seed

Abstract Attachment of Enterobacter cloacae EcCT-50,—a biological seed protectant used to control the seed-rotting fungi, Pythium ultimum—to cotton seed was examined using conventional fluorescent microscopy (CFM), scanning electron microscopy (SEM), and laser scanning microscopy (LSM). In sand microcosms, E. cloacae quickly attached to the seed coat, with maximum attachment, 3 to 5 h after inoculation at 24°C. In contrast, initial attachment of non-bacterized seed by Pythium ultimum was not observed until 6 h (and not until 8 h on bacterized seeds). Comparison of the movement of E. cloacae and P. ultimum in seed exudate gradient semi-soft agar showed faster movement by the bacterium within the first 6 h, and reduction of P. ultimum hyphal and germ tube growth in the presence of the bacterium. Microscopic observation of the seed coat revealed an early, intimate association, mediated, in part, by fimbriae, and confirmed a loose association of E. cloacae with the seed coat previously reported. Spatially, the attached E. cloacae cells were distributed over the entire surface of the seed coat, but were especially abundant in the groves and near cracks where water imbibition and seed exudate release may occur. As the seed germinated and exposed various seed tissues, the bacterium rapidly attached to these tissues. Attachment of the bacterium to the surface of intact germinating seeds, excised seed coat, polystyrene, and glass was 300, 110, 51, and <1 cell field⁻¹ 3 h⁻¹, respectively, suggesting that attachment is enhanced by seed germination. Attachment of E. cloacae to the seed coat was optimum in sands with high water concentrations, at temperatures of 18 to 30°C, and at times that corresponded with optimum water imbibition during germination. Using several assays, attachment was shown to be enhanced by seed exudate, and compounds such as methanol, fructose, and calcium. The results suggest that the release of certain nutrients and water imbibition during germination may play a role in the rapid attachment to the seed by E. cloacae. The ability of E. cloacae to rapidly move and attach to the seed coat may be related to its ability to function as a biocontrol agent. Received: 24 April 1997; Accepted 29 October 1997     

Proteomic Profiling of the Aleurone Layer of Mature Arabidopsis thaliana Seed

The aleurone layer of mature Arabidopsis thaliana seed plays important roles in seed germination and dormancy. However, the proteomic profile of this cell layer is unknown partly because it is difficult to separate this thin cell layer from the mature seeds. In this study, we have used a simple technique to separate the aleurone layer along with the seed coat following germination of seeds and determined for the first time the putative protein composition of this cell layer. By subjecting the total proteins extracted from the seed coat to 2D gel electrophoresis followed by liquid chromatography/tandem mass spectrometry, we identified four AGI loci, AT4G28520, AT5G44120, AT1G03880, and AT1G03890; all of which belong to the seed storage family of proteins. Because in Arabidopsis the diploid aleurone cells of the seed coat perform protein storage functions similar to that of triploid endosperm of other plant species, it is assumed that the above AGI loci are associated with the aleurone layer of the seed coat.     

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.     

Proteomic analysis of rice embryo: an approach for investigating Galpha protein-regulated proteins

The rice dwarf1 (d1) mutant, which lacks the alpha subunit of a heterotrimeric G protein (Galpha protein), shows abnormal morphology due to shortened internodes, dark green leaves and grains that are small and round. Proteome analysis was used in this study to aid in determining the function of Galpha protein in rice embryos. Using 2-DE, seven seed embryo proteins were shown to be down-regulated in the d1 mutant as compared with its wild type. These seven proteins included a receptor for activated C-kinase (RACK) and six rice embryo globulin-2 proteins (REG2). The six REG2 have similar molecular masses with minor differences in pI. In addition to the reduced accumulation of RACK in the d1 mutant, the increase in QL/d1, in which a constitutively active form of the Galpha protein is expressed, was significantly higher as compared with wild type. The level of accumulation of these seven proteins during seed development and maturation did not change significantly until the 2nd wk after pollination. Reduced accumulation of these seven proteins started in the d1 mutant at the 3rd wk after pollination, and continued until seed maturation was complete. All seven proteins were completely absent 24 h after imbibition in both d1 mutant and its wild type. However, the phytohormone abscisic acid promoted the expression level of RACK after imbibition in the wild type as compared with d1 mutant. These results suggest that RACK is regulated by Galpha-protein and plays an important role in a basic cellular process as well as in rice embryogenesis and germination.     

Seed coats: Structure,development, composition,and biotechnology

Summary Although seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be examined from the perspective of molecular genetics and control of development. The seed coat, plays a vital role in the life cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the seed coat are a number of unique tissues that undergo differentiation to serve specific functions in the seed. A large number of genes are known to be specifically expressed within the seed coat tissues; however, very few of them are understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse ways, including defense and control of development. Many of the compounds are sources of industrial products and are components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat development and composition. In this review, we will examine the recent advances in seed coat, biology from the perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities for seed coat modifications in the future. The authors have contributed equally and are considered first authors.     

Proteome analysis of embryo and endosperm from germinating tomato seeds

Proteome analysis of embryo and endosperm tissues from germinating tomato seed was conducted using 1-DE, 2-DE, and MS. Mobilization of the most abundant proteins, which showed similar profiles in the two tissues, occurred first in the endosperm. CBB R-250 staining of 2-DE gels revealed 352 and 369 major protein spots in the embryo and endosperm, respectively, at 0 h. Of these, 75 major spots were selected, excised, in-gel digested with trypsin, and analyzed by MALDI-TOF-MS and/or LC-ESI-Q/TOF-MS/MS. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 47 proteins identified. Embryo-specific proteins included a BAC19.13 homologue, whereas four proteins specific to the endosperm were tomato mosaic virus coat proteins related to defense mechanisms. The most abundant proteins both in the embryo and endosperm were seed storage proteins, i.e., legumins (11 spots), vicilins (11 spots), albumin (2 spots). Housekeeping enzymes, actin-binding profilin, defense-related protein kinases, nonspecific lipid transfer protein, and proteins involved in general metabolism were also identified. The roles of some of the proteins identified in the embryo and endosperm are discussed in relation to seed germination in tomato.     

小麦种子成熟和萌发过程中的假萌发素活性

用SDS-PAGE方法研究了假萌发素(ψG)在小麦种子成熟和萌发过程中活性的变化.结果表明:在种子成熟过程中只有ψG表达,扬花后10 d,在颖壳、内外桴、种皮和果皮中皆可检测到ψG的草酸氧化酶活性,随着发育进程的推进,ψG的活性增大.在种子萌发过程中,在小麦品种中育5号的维管束过渡区中除了萌发素G和G'外,还可检测到ψG的草酸氧化酶活性.由于ψG在种子成熟过程中主要存在于颖壳、内外桴、果皮及种皮这些保护组织中,且开始大量表达的时间正是生长接近停止时,于是推测ψG很可能通过降解草酸产生H2O2而推动这些组织细胞壁的木质化.