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.     

Repetitive proline-rich proteins in the extracellular matrix of the plant cell

Several classes of hydroxyproline-rich proteins have been found in the cell walls of plants. Monomeric forms of the proteins can be solubilized from the walls, but the majority of the proteins are insolubilized by as yet unidentified crosslinks. The proteins have repeated sequences and are often rich in basic amino acids. The repeat proline-rich region may be serving to generate an elongated rod-like structure while the regularly spaced lysines probably interact with the acidic pectins. Several of the genes coding for these proteins have been isolated, and their expression has been found in some cases to be tissue specific and in others inducible by hormones and various types of stress.     

Hourglass cell development in the soybean seed coat

Background and Aims

Hourglass cells (HGCs) are prominent cells in the soybean seed coat, and have potential use as ‘phytofactories’ to produce specific proteins of interest. Previous studies have shown that HGCs initiate differentiation at about 9 d post-anthesis (dpa), assuming their characteristic morphology by 18 dpa. This study aims to document the structural changes in HGCs during this critical period, and to relate these changes to the concurrent development of a specific soybean peroxidase (SBP) encoded by the Ep gene.

Methods

Pods were collected from plants at specific growth stages. Fresh material was processed for analysis of Ep peroxidase activity. Tissues were processed for scanning and transmission electron microscopy, as well as extracted for western blotting. A null variety lacking expression of Ep peroxidase was grown as a control.

Key Results and Conclusions

At 9 dpa, HGCs are typical undifferentiated plant cells, but from 12–18 dpa they undergo rapid changes in their internal and external structure. By 18 dpa, they have assumed the characteristic hourglass shape with thick cell walls, intercellular air spaces and large central vacuoles. By 45 dpa, all organelles in HGCs have been degraded. Additional observations indicate that plasmodesmata connect all cell types. SBP activity and SBP protein are detectable in the HGC before they are fully differentiated (approx. 18 dpa). In very early stages, SBP activity appears localized in a vacuole as previously predicted. These results increase our understanding of the structure and development of the HGC and will be valuable for future studies aimed at protein targeting to components of the HGC endomembrane systems.     

The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus)

The seeds of the Jack bean, Canavalia ensiformis (L) DC are known to contain several toxic substances that prevent their utilisation as food for humans and animals. The lectin concanavalin A and the enzyme urease are the best known of these proteins. We have found that many proteins present in the seeds of the Jack bean, like trypsin inhibitors and canatoxin, are detrimental to the development of the bruchid insect Callosobruchus maculatus (F) (Coleoptera: Bruchidae). Among these proteins, canavalin (vicilin, 7S globulin) was found to be expressed in the seed coat. We suggest that seed coat canavalin, in addition to other detrimental proteins expressed in this tissue, may have been of importance in the evolutionary discrimination of the seeds of this legume by non-pest bruchids.     

Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)

Soybean seed coat peroxidase (SBP) is a valuable enzyme having a broad variety of applications in analytical chemistry, biochemistry, and food processing. In the present study, the sscp gene (Gene ID: 548068) was optimized based on the preferred codon usage of Escherichia coli, synthesized, and expressed in E. coli BL21(DE3). SDS-PAGE and western blot analysis of this expressed protein revealed that its molecular weight is approximately 39?kDa. The effects of induction temperature, concentration of isopropyl-β-D-thiogalactoside and hemin, induction time, expression time were optimized to enhance SBP production with a maximum activity of 11.23?U/mL (8.64?U/mg total protein). Furthermore, the kinetics of enzyme-catalyzed reactions of recombinant protein was determined. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as substrate, optimum reaction temperature and pH of the enzyme were 85°C and 5.0, respectively. The effects of metal ions on the enzymatic reaction were also further investigated. The SBP was successfully expressed in E. coli BL21(DE3) which would provide a more efficient production strategy for industrial applications of SBP.     

果皮和种皮微形态特征在杜鹃花属系统学研究中的应用

采用扫描电子显微镜对国产杜鹃花属Rhododendron6个亚属的代表种和近缘类群杜香属Ledum杜香L.palustre的果皮和种皮微形态特征进行观察,对杜鹃花属植物果皮微形态特征进行了系统描述,并通过比较现存杜鹃花属植物和种子化石,新发现一些居间的种子类型。结果表明,果皮和种皮微形态特征具有一定的系统学意义。叶状苞亚属subgen.Therorhodion的叶状苞杜鹃R.redowskianum的果实有短而疏的刺毛,无气孔,种子为无翅类,扁平,外围轮廓长椭圆形。杜鹃亚属subgen.Rhododendron植物果皮为百合花杜鹃型,具有鳞片,气孔器散生于指状突起之间,与叶表皮的微形态特征一致,其种子为百合花杜鹃型,表面具宽而浅的沟,呈脑纹状,有别于无鳞类杜鹃花。常绿杜鹃亚属subgen.Hymenanthes果皮为云锦杜鹃型,其角质层表面不规则,无表皮毛,偶见气孔;种子为云锦杜鹃型。映山红亚属subgen.Tsutsusi果皮为岭南杜鹃型,密生长表皮毛,角质层致密;种子为南边杜鹃型和岭南杜鹃型。微形态特征与“常绿杜鹃亚属和(国产)映山红亚属均为内部支持率很高的单系类群”的分子系统发育研究结果一致。马银花亚属subgen.Azaleastrum的马银花组sect.Azaleastrum和长蕊杜鹃组sect.Choniastrum微形态区别明显,支持各自为独立的单系类群。羊踯躅亚属subgen.Pentanthera的羊踯躅R.molle果皮特征明显,可与其他亚属区分,但种子类型更接近常绿杜鹃亚属。本研究结合分子系统发育资料和叶表皮微形态特征讨论了一些近缘类群的系统发育关系;研究结果支持将腺萼马银花处理为独立的种。     

Insecticidal activities of Ginkgo biloba seed coat extracts against Spodoptera exigua (Lepidoptera: Noctuidae)

The present study relates to a methanol extract of the seed coat of Ginkgo biloba, and tested particularly on the third instar larvae of Spodoptera exigua. The extract was found to have an inhibitory effect on the growth of the larvae besides bringing a change in the nutrient reserves in the body of the insect. Topical application of five different doses of the methanol extract resulted in a mortal effect to third instar larvae of S. exigua that is very much dependent on the dose as well as duration of exposure. Lower doses revealed lower mortality after 24 h of application. At doses of 1.00, 2.00, 4.00, 8.00 and 16.00 ng/larva, mortalities were 9.25, 26.07, 50.32, 56.28 and 92.44%, respectively. The dose for 50% mortality (LD₅₀) of methanol extracts by applied by a topical method with 1 µL of acetone solution was 1.92 ng/larva. Nutrient reserves like protein, glycogen and lipid are known to regulate pupation and adult emergence. These reserves have been found to be lower in treated larvae, indicating the insecticidal role of methanol extracts from G. biloba against third instar larvae of S. exigua.     

The structure of the seed in some Western Australian species of the genus Hibbertia (Dilleniaceae)

The internal structure of the seed was studied for four Western Australian species of the genus Hibbertia (Fam.: Dilleniaceae). The principal mechanical layer of the seed coat is the endotesta. The large cells of the endotesta are heavily cutinized in potentially viable seeds of all examined species. However, results for H. hypericoides show that the cells of the endotesta are distinctly less cutinized in non-viable seeds. The tegmen consists of only one layer which is spirally thickened. Remains of the nucellus enclose the endosperm. The endosperm is rich in both starch and lipids, and the embryo is underdeveloped and minute.     

Characterization of the uptake of sucrose and glucose by isolated seed coat halves of developing pea seeds. Evidence that a sugar facilitator with diffusional kinetics is involved in seed coat unloading

Uptake of ¹⁴C-labelled sucrose and glucose by isolated seed coat halves of pea (Pisum sativum L. cv. Marzia) seeds was measured in the concentration range <0.1 μM to 100 mM. The initial influx of sucrose was strictly proportional to the external concentration, with a coefficient of proportionality (k) of 6.2 μmol·(g FW)^?1·min^?1·M^?1. Sucrose influx was not affected by 10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), but it was inhibited by 40% in the presence of 2.5 mM p-chloromercuribenzenesulfonic acid (PCMBS). Influx with diffusional kinetics was also observed for glucose (k = 4.8 μmol·(g FW)^?1·min ^?1·M ^?1) and mannitol (k = 5.1 μmol·(g FW)^?1·min^?1·M^?1). For glucose an additional saturable system was found (K_m = 0.26 mM, V _max = 4.2 nmol·(g FW)^?1·min^?1), which appeared to be completely inhibited by CCCP and partly by PCMBS. In contrast to the diffusional pathway, uptake by this saturable system was slightly pH-dependent, with an optimum at pH 5.5. The influx of sucrose appears to be by the same pathway as the efflux of endogenous sucrose, which was inhibited by 36% in the presence of 2.5 mM PCMBS (De Jong A, Wolswinkel P, 1995, Physiol Plant 94: 78–86). It is argued that passive transport may be the only mechanism for sucrose transport through the plasma membrane of seed coat parenchyma cells. The estimated permeability coefficient of the plasma membrane for sucrose (P = 3.5·10^?7 cm·s^?1) is more than 1 × 10⁶-fold higher than that reported for artificial lipid membranes. This relatively high permeability is hypothesized to result from pore-forming proteins that allow the diffusion of sucrose. Furthermore, it is shown that a sucrose gradient across the plasma membrane of the seed coat parenchyma of only 22 mM will suffice to result in the net efflux of sucrose which is required to feed the embryo.     

Specificity in the immobilisation of cell wall proteins in response to different elicitor molecules in suspension-cultured cells of French bean (Phaseolus vulgaris L.)

A characteristic of the defence response is the immobilisation of wall proteins possibly through the formation of covalent cross-links and the subsequent barrier formation against pathogens. A requirement for this is the generation of active oxygen species, particularly hydrogen peroxide. In the present work, we examine in depth the requirement for H₂O₂ and the specificity of the immobilisation with respect to particular wall proteins. Salt-extractable wall proteins were analysed for hydroxyproline content and the subset of proteins with this post-translational modification was found to be small. About 50 proteins were found to be easily salt-extractable and in response to elicitor treatment about 5 were found to be specifically immobilised. Immobilisation was very rapid and completed within 15 min after elicitation, and dependent upon the type of elicitor and the intensity of the production of active oxygen species. N-terminal sequencing and amino acid analysis revealed that, apart from one polypeptide, all immobilised proteins were (hydroxy)proline-containing glycoproteins with O-linked oligosaccharide side chains. In contrast, N-linked glycoproteins were not immobilised. N-terminal protein sequencing revealed the immobilised HRGPs to be novel, but both extensin and PRP-like. Implications of these findings for both pathogenic and symbiotic processes are also discussed.     

The endotegmen tuberculae: an account of little-known structures from the seed coat of the Hydrocharitoideae (Hydrocharitaceae) and Najas (Najadaceae)

SHAFFER-FEHRE, M., 1991. The endotegmen tuberculae: an account of little-known structures from the seed coat of the Hydrocharitoideae (Hydrocharitaceae) and of Najas (Najadaceae). The sub-cellular protuberances from the inner tegmen layer have been given various names in earlier literature. Here named 'endotegmen tuberculae', they vary in shape, size and density of distribution in the endotegmen of different taxa. These characteristics make them useful for taxonomic research at both genus and species level. Their shape is a major consideration in transferring Hydrocharis dubia to the genus Limnobium. A further structure, 'exotegmen tuberculae', has been observed for the first time. This paper gives a survey of these structures and illustrates their taxonomic importance and its implications.     

A taxometric analysis of seed proteins in the genusBriza s. l. (Poaceae)

The species ofBriza L. s. l. are disjunctly distributed between Eurasia and South America. A taxometric analysis has been carried out, using data from electrophoresis of seed polypeptides and published morphological characters. Evidence from a cluster analysis of seed polypeptide data strongly supports the recognition of two different natural groups, a S. American group and a Eurasian group, and is consistent with karyotype data, but does not support the generic groupings of South American taxa derived from the morphological data.     

Effect of the seed coat on water uptake and electrolyt leakage of sorghum (Sorghum bicolor (L.) Moench) seeds

Four seed lots of sorghum cultivar IS 34213 were obtained which had been harvested early or at full maturity and then dried either swiftly in the sun or slowly in the shade. The earlier harvested seeds were lighter, smaller and of lower density. Earlier harvested seeds had pericarps which permitted faster water uptake and greater leakage of electrolytes. Water uptake and electrolyte leakage were positively correlated. The method of seed drying had no effect on water uptake or leakage. Greater leakage was associated with lower final germination. The results are interpreted in relation to germination testing.     

Isolation and characterization of a galactorhamnan polysaccharide from the seed coat of Canavalia ensiformis that is toxic to the cowpea weevil (Callosobruchus maculatus)

We have isolated a water-soluble polysaccharide from the Jack bean [Canavalia ensiformis (L) DC] seed coat that was shown to be highly detrimental to larval development of the cowpea weevil (Callosobruchus maculatus) (Coleoptera: Bruchidae). Determination of the composition and structure of this polysaccharide showed that it is a galactorhamnan with an M_w of 883.0, containing 92% rhamnose and 8% galactose. The polymer is formed by a main chain of rhamnose (1to2) substituted at O-4 by galactose nonreducing end-units. Immunolocalisation by light and electron microscopy showed that this polysaccharide is localised in the innermost cell layer of the seed coat and also in cotyledon tissues in the cytoplasm space. The presence of this toxic polysaccharide in the testa of a non-host seed may have been important for the evolutionary discrimination of legume seeds by bruchids.     

不同培养基上繁殖的昆虫病原线虫格氏线虫表皮蛋白的差异

表皮蛋白(surface coat proteins, SCPs)是格氏线虫Steinernema glaseri克服寄主免疫系统的关键因素, 能够抑制昆虫免疫反应, 促进线虫的侵染。为了进一步研究表皮蛋白抑制昆虫免疫的作用机理和线虫与寄主间的相互关系, 我们比较分析了不同培养基繁殖的格氏线虫表皮蛋白的差异。我们用人工培养基和大蜡螟Galleria mellonella末龄（5龄）幼虫分别培养得到格氏线虫侵染期幼虫, 利用酒精提取表皮蛋白。SDS-PAGE和非变性PAGE分析显示, 大蜡螟来源的格氏线虫的表皮蛋白具有更多的蛋白条带。体外和体内的裂解血细胞实验表明, 大蜡螟来源的格氏线虫的表皮蛋白表现出强烈的裂解血细胞活性, 而人工培养基来源的格氏线虫的表皮蛋白活力不明显; 且具有裂解血细胞活性的表皮蛋白为诱导表达型蛋白。不同培养基来源的格氏线虫的表皮蛋白组成的差异和活性的不同, 表明格氏线虫表皮蛋白的产生受线虫培养条件影响较大。     

Development and structure of the seed of Ozoroa paniculosa (Anacardiaceae) and taxonomic notes

The anatropous, bitegmic and crassinucellar ovule has a nuclear endosperm development. It is further characterized by a hypostase sensu lato. This hypostase being an integral part of the chalaza undergoes a secondary extension with it. At maturity the exalbuminous seed is partially pachychalazal and therefore two anatomically distinct larger parts can be distinguished in the mature seed coat. An endotegmen typifies the integumentary seed coat, while a saddle-shaped hypostase characterizes the chalazal seed coat. This seed coat shows several characteristics of the typical anacardiaceous pachychalazal seed. The cotyledons store lipids and protein as nutrient reserveS. A well-developed cuticle, cuticular layer, cutin and callose in the hypostase cell walls, as well as tannin-like deposits in the seed coat, protect the physiologically ripe seed against dehydration.