Tissue- and stage-specific expression of a soybean (Glycine max L.) seed-maturation,biotinylated protein

A cDNA clone GmPM4 which encodes mRNA species in mature or dry soybean seeds was characterized. DNA sequence analysis shows that the deduced polypeptides have a molecular mass of 68 kDa. GmPM4 proteins have a relatively high amino acid sequence homology with a major biotinylated protein isolated from pea seeds, SBP65, but both of these proteins differ markedly from that of presently known biotin enzymes. The accumulation of GmPM4 mRNA is detectable in the leaf primodium and the vascular tissues of the hypocotyl-radicle axis of mature seeds, and the GmPM4 proteins are present at high levels in dry and mature soybean seeds, but not in fresh immature seeds. It degrades rapidly at the early stage of seed germination. These proteins are boiling-soluble and biotinylated when they are present endogenously in soybean seeds; however, the same recombinant protein expressed in Escherichia coli is boiling-soluble, but it is not biotinylated.     

Centrifugal partition chromatography as a tool for preparative purification of pea albumin with enhanced yields

A new procedure including the use of centrifugal partition chromatography (CPC) is proposed to purify PA1b and its isoforms. These pea (Pisum sativum L.) seed proteins are toxic against weevils and can be used as an environment-friendly insecticide. CPC was applied to a whole albumin fraction prepared from pea flour. The butanol:aqueous TFA system used in CPC allowed the separation of PA1b from other albumins and a degree of purification above 95%. Compared to analytical procedures based on methanol extraction, anion exchange and then reversed-phase chromatography (RPC), CPC recovered PA1b in much better yield, which is indispensable for large-scale purification of a biodegradable insecticide.     

Differential expression of a chlorophyll a/b binding protein gene and a proline rich protein gene in juvenile and mature phase English ivy (Hedera helix)

Two cDNA clones representing mRNAs which are differentially expressed during in vitro culture of juvenile and mature leaf petioles of English ivy ( Hedera helix L.) were isolated by differential screening. The mRNA represented by clone HW101 is expressed at a higher level in untreated juvenile than in untreated mature in-vitro-cultured petioles. Treatment of petioles with α-naphthaleneacetic acid (NAA) at the initiation of culture decreases HW101 mRNA levels in juvenile but not mature, petioles. In intact plants. HW101 mRNA is expressed at a higher level in juvenile laminae, petioles and stems than in identical tissues of mature plants. DNA sequence analysis indicates that HW1O1 cDNA is significantly similar to a light harvesting chlorophyll a/b binding protein gene ( Lhcb ) of pea. The gene represented by the second clone. HW103, is expressed at a higher level in mature than in juvenile in-vitro-cultured petisoles. Treatment of petioles with NAA at the initiation of culture decreases HW103 mRNA levels in chronologically young mature but not older mature and juvenile petioles. However, expression of the HW103 gene is not detectable in petioles, or in any other vegetative organ tested, immediately after excision. It is, however, expressed in developing seeds. In otherwise intact plants, the HW103 gene is expressed in wounded petioles of mature plants 5 days after wounding but not in wounded petioles of juvenile plants. It is also expressed at a higher level in wounded stems of mature plants than in those of juvenile plants. However, it is not expressed in wounded lamina of either juvenile or mature plants. DNA sequence analysis indicates that HW103 cDNA is similar to a cell wall proline rich protein (PRP) gene of soybean. This is the first report of differential expression of a PRP gene in tissues from juvenile and mature plants. Southern blot analysis of nuclear DNA of H. helix shows that both HW101 and HW103 are members of small gene families.     

Albumin storage proteins in the protein bodies of castor bean

Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of closely related albumins localized in the matrix of the organelle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds.     

Genetic heterogeneity in propionic acidemia patients with alpha-subunit defects. Identification of five novel mutations, one of them causing instability of the protein

The inherited metabolic disease propionic acidemia (PA) can result from mutations in either of the genes PCCA or PCCB, which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl CoA-carboxylase. In this work we have analyzed the molecular basis of PCCA gene defects, studying mRNA levels and identifying putative disease causing mutations. A total of 10 different mutations, none predominant, are present in a sample of 24 mutant alleles studied. Five novel mutations are reported here for the first time. A neutral polymorphism and a variant allele present in the general population were also detected. To examine the effect of a point mutation (M348K) involving a highly conserved residue, we have carried out in vitro expression of normal and mutant PCCA cDNA and analyzed the mitochondrial import and stability of the resulting proteins. Both wild-type and mutant proteins were imported into mitochondria and processed into the mature form with similar efficiency, but the mature mutant M348K protein decayed more rapidly than did the wild-type, indicating a reduced stability, which is probably the disease-causing mechanism.     

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post‐translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.     

PA1b, an insecticidal protein extracted from pea seeds (Pisum sativum): 1H-2-D NMR study and molecular modeling

PA1b (pea albumin 1, subunit b) is a 37-amino acid cysteine-rich plant defense protein isolated from pea seeds (Pisum sativum). It induces short-term mortality in several pests, among which the cereal weevils Sitophilus sp. (Sitophilus oryzae, Sitophilus granarius, and Sitophilus zeamais) that are a major nuisance for stored cereals, all over the world. As such, PA1b is the first genuine protein phytotoxin specifically toxic to insects, which makes it a promising tool for seed weevil damage control. We have determined the 3-D solution structure of PA1b, using 2-D homonuclear proton NMR methods and molecular modeling. The primary sequence of the protein does not share similarities with other known toxins. It includes six cysteines forming three disulfide bridges. However, because of PA1b resistance to protease cleavage, conventional methods failed to establish the connectivity pattern. Our first attempts to assign the disulfide network from NOE data alone remained unsuccessful due to the tight packing of the cysteine residues within the core of the molecule. Yet, the use of ambiguous disulfide restraints within ARIA allowed us to establish that PA1b belongs to the inhibitor cystine-knot family. It exhibits the structural features that are characteristic of the knottin fold, namely, a triple-stranded antiparallel beta-sheet with a long flexible loop connecting the first to the second strand and a series of turns. A comparison of the structural properties of PA1b with that of structurally related proteins adopting a knottin fold and exhibiting a diverse range of biological activities shows that the electrostatic and lipophilic potentials at the surface of PA1b are very close to those found for the spider toxin ACTX-Hi:OB4219, thereby suggesting activity on ion channels.     

Changes in Polysomes and RNA in Developing Seeds of Chickpea (Cicer arietinum L.)

In the present investigation changes in polyribosomes and RNAs in the developing seeds of chickpea (Cicer arietinum L.) have been studied. The total polysome yield was higher in the early stages of development and declined at the later stages. The maximum level of polyribosomes was obtained at 18 days after flowering and a drastic decrease was noticed at maturity. The total RNA yield correlated with the polysomal yield. Northern hybridization with a heterologous probe (pea legumin cDNA) gave distinct hybridization with the mRNA coding for legumin proteins at different stages of seed development. Hybridization showed a direct relation between mRNA levels and seed weight accumulation.     

Soybean vegetative storage protein structure and gene expression

Depodded soybean (Glycine max [L] Merr. cv Williams) plants accumulate high levels of a glycoprotein in their leaves that has many features of a storage protein. The protein is found in all vegetative tissues which have been examined but not in the seeds. Translation in vitro indicated that elevated mRNA levels were at least partially responsible for the specific increase in vegetative storage protein. cDNA clones were isolated and sequenced, and an amino acid sequence was predicted. Although the amino acid composition is similar to that of seed storage proteins, no sequence similarity could be detected. Northern blot hybridization confirmed a large increase in vegetative storage protein mRNA in leaves of depodded plants. The vegetative storage proteins are represented by about four gene copies in the haploid genome.     

cDNA and protein sequence of a major pea seed albumin (PA 2 : Mr≈26 000)

Summary Pea albumin 2 (PA2:M_r26000) is a major component of the albumin fraction derived from aqueous salt extracts of pea seed. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and chromatography on DEAE-Sephacel resolve PA2 into two closely related components (PA2a and PA2b). A cDNA clone coding for one of these components has been sequenced and the deduced amino acid sequence compared with partial, chemically-determined sequences for cyanogen bromide peptides from both PA2 components. Complete amino acid sequences were obtained for the C-terminal peptides. The PA2 molecule of 230 amino acids contains four imperfect repeat sequences each of approximately 57 amino acids in length.The combined sequence data, together with a comparison of PA2-related polypeptides produced in vitro and in vivo, indicate that PA2 is synthesized without a signal sequence and does not undergo significant post-translational modification. Although both forms of PA2 contain Asn-X-Thr consensus sequences, neither form is glycosylated. Accumulation of PA2 contributes approximately 11% of the sulfur-amino acids in pea seeds (cysteine plus methionine equals 2.6 residues percent). Suppression of levels of PA2 polypeptides and their mRNAs in developing seeds of sulfur-deficient plants is less marked than that for legumin, in spite of the lower content of sulfur-amino acids in legumin.     

Divergence of the two albumins of X. laevis. Evidence for the glycosylation of the major 74K albumin

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis, differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Divergence of the Two Albumins of X. laevis

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis , differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Characterization of an apoplastic basic thaumatin-like protein from recalcitrant chestnut seeds

Mature chestnut seeds, with one of the highest moisture contents described to date, accumulate certain defensive proteins at unusually elevated levels. In this work a major 23-kDa thaumatin-like protein, termed CsTL1, has been purified from mature chestnut ( Castanea sativa ) cotyledons. Amino acid sequencing and characterization of its full-length cDNA indicate that CsTL1 is synthesized as a preprotein with a signal peptide 22 amino acids in length. The mature protein contains 16 conserved cysteine residues presumably involved in disulfide bonding and has a high isoelectric point (ca. 9). Unlike most basic pathogenesis-related (PR) proteins, mature CsTL1 is localized to the extracellular matrix, as revealed by immunoelectron microscopy studies of cotyledonary cells. The isolated protein has in vitro antifungal activity against Trichoderma viride and Fusarium oxysporum and shows strong synergistic effects with CsCh1, the most abundant chestnut cotyledon endochitinase. Moreover, both CsTL1 and CsCh1 appear to be regulated in the same manner during seed development and germination. These observations, along with the recent finding of endoglucanase activity for some TL proteins, support the notion that CsTL1 and CsCh1 are part of a complex seed defensive system against microbial growth. Another possibility is that these, and probably other seed PR proteins, have antifreeze activity. Both functions would be particularly relevant for chestnut seeds given their remarkable moisture content at maturity.