An antifungal peptide from passion fruit (Passiflora edulis) seeds with similarities to 2S albumin proteins

An actual worldwide problem consists of an expressive increase of economic losses and health problems caused by fungi. In order to solve this problem, several studies have been concentrating on the screening of novel plant defence peptides with antifungal activities. These peptides are commonly characterized by having low molecular masses and cationic charges. This present work reports on the purification and characterization of a novel plant peptide of 5.0 kDa, Pe-AFP1, purified from the seeds of passion fruit (Passiflora edulis). Purification was achieved using a Red-Sepharose Cl-6B affinity column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Pe-AFP1 was able of inhibiting the development of the filamentous fungi Trichoderma harzianum, Fusarium oxysporum, and Aspergillus fumigatus with IC50 values of 32, 34, and 40 microg ml(-1), respectively, but not of Rhyzoctonia solani, Paracoccidioides brasiliensis and Candida albicans. This protein was also subjected to automated N-terminal amino acid sequence, showing high degree of similarities to storage 2S albumins, adding a new member to this protein-defence family. The discovery of Pe-AFP1 could contribute, in a near future, to the development of biotechnological products as antifungal drugs and transgenic plants with enhanced resistance to pathogenic fungi.     

Characterization of 2S seed storage protein of Brassica campestris and its antigenic homology with seed proteins of other Cruciferae.

The low molecular weight seed storage protein of Brassica campestris has been isolated and its amino acid composition determined. Antibody raised against this low molecular weight protein has been used to compare the antigenic similarity between the low molecular weight storage proteins of different Cruciferae seeds by immunoprecipitation and Western blotting. These studies revealed the existence of antigenically homologous proteins of identical molecular weights in seeds of other Cruciferae but absent in some other dicots like mung bean and tobacco seeds.     

Characterization of a soybean leaf protein that is related to the seed lectin and is increased with pod removal

Levels of several polypeptides in addition to the vegetative storage protein (VSP) increase in soybean leaves following depodding. Two of these polypeptides interact specifically with antibodies raised against the seed lectins of Phaseolus vulgaris and soybean. The two polypeptides, which had apparent molecular masses of 29,000 daltons and 33,000 daltons, were present in the sink-deprived plants but not in control podded plants and were the subunit polypeptides of a glycoprotein designated lectin-related protein (LRP). Soybean LRP was purified to near homogeneity by a combination of ammonium sulfate precipitation and gel filtration. Dialysis of the resuspended ammonium sulfate precipitate caused LRP to reprecipitate, and LRP was soluble only in the presence of molar NaCl. The native relative molecular mass of LRP was 119,000 daltons, a size consistent with a tetrameric organization of the two polypeptides. LRP precipitated during dialysis in association with a 28,000 dalton polypeptide. The protein coprecipitating with LRP was identified as the dimer of the 28,000 dalton subunit of VSP, one of three native isomeric forms of VSP occurring in leaves of depodded plants. Although the specific association between LRP and VSP was intriguing, an in vivo interaction between LRP and VSP was doubtful. LRP was shown to be immunologically similar to soybean agglutinin but did not have detectable hemagglutinating activity. LRP also was shown to be made up of polypeptides distinct from soybean agglutinin.     

Characterization of matteuccin, the 2.2S storage protein of the ostrich fern. Evolutionary relationship to angiosperm seed storage proteins

The 2.2S spore storage protein (matteuccin) of the ostrich fern, Matteuccia struthiopteris, has been isolated and characterized. It is a small basic protein consisting of two disulfide-linked polypeptides with approximate molecular masses of 3.0 kDa and 8.0 kDa. At least four different isoforms exist where two of the forms differ from the other by having a slightly smaller heavy chain. Amino acid analysis reveals that the 2.2S protein is rich in arginine. Almost complete amino acid sequence information was obtained for the light chain and a partial sequence for the heavy chain. Amino acid sequence comparison reveals that this protein shows a high similarity to seed storage proteins in different angiosperm species in spite of the fact that the common ancestor of ferns and angiosperms lived more than 300 million years ago.     

Characterization of a Saponaria officinalis seed ribosome-inactivating protein: immunoreactivity and sequence homologies

A ribosome inactivating protein from Saponaria officinalis, SO-6, was purified and the N-terminus sequenced. The sequence shows extensive homology with Pokeweed antiviral protein, Pokeweed antiviral protein II, Pokeweed antiviral seed protein and dodecandrin. SDS gel electrophoresis in the Laemmli system revealed two bands of similar intensities with a smear between them, probably an artifact due to the high pI of the protein. Use of a harsher denaturing gel system resulted in one band in electrophoresis. Immune antisera was raised in rabbits against this protein and it cross reacted with other proteins (SO-5, SO-8 and SO-9) from seeds of Saponaria officinalis, but not with gelonin, Momordica charantia inhibitor and dianthin 32.     

Potato lectin: a modular protein sharing sequence similarities with the extensin family, the hevein lectin family, and snake venom disintegrins (platelet aggregation inhibitors)

Potato (Solanum tuberosum) lectin, is a chimeric chitin-binding protein comprised of a lectin domain fused to a hydroxyproline-rich glycoprotein domain. Here peptide sequence information from both domains is presented. A partial sequence of a major tryptic peptide T2: Leu-Pro-Ser-Hyp-Hyp-Hyp-Hyp-Hyp-Hyp-(His)-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Hyp- was similar to the ‘P3’ type extensin major repetitive sequence: Ser-Hyp-Hyp-Hyp-Hyp-Ser-Hyp-Ser-Hyp-Hyp-Hyp-Hyp-suggesting common evolutionary origins for the extensins and the hydroxyproline-rich glycoprotein (HRGP) domain of potato lectin. Furthermore, alignment of three chymotryptic peptides from potato lectin, C1: Cys-Gly-Thr-Thr-Ser-Asp-Tyr, C2: Cys-Ser-Pro-Gly-Tyr, and C8: Thr-Gly-Glu-Cys-Cys-Ser-Ile with similar sequences from the hevein lectin family indicates that they have homologous chitin-binding domains, and hence have common evolutionary origins. Finally, all plant chitin-binding domains examined bore a remarkable sequence similarity, particularly in the spacing of Cys residues, to the disintegrins (platelet aggregation inhibitors) which occur in crotalid and viperid snake venoms. As such, sequence similarities not only identify potato lectin as a member of both the hevein and extensin families of plant proteins, but also suggest that an archetypal polypeptide module gave rise to both the plant chitin-binding domain and the reptile disintegrins.     

Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

A fern spore storage protein is genetically similar to the 1.7 S seed storage protein ofBrassica napus

The ostrich fern, Matteuccia struthiopteris L., contains two globulin spore storage proteins of 2.2 S and 11.3 S, with physical characteristics similar to those of seed storage proteins of Brassica napus (rapeseed) and Raphanus sativus (radish). By the use of a cloned cDNA that encodes the 1.7 S B. napus storage protein (napin), gene sequences that hybridized with napin were detected in fern nuclear DNA, and a 900-nucleotide homologous mRNA was detected in developing spores. In vitro translation of this fern mRNA produced a 22-kD polypeptide comparable in size to the 21-kD precursor polypeptide identified in Brassica. No hybridizations were observed between the Brassica 12 S clone and either fern DNA or developing spore mRNA.     

Solution structure of RicC3, a 2S albumin storage protein from Ricinus communis

The three-dimensional structure in aqueous solution of recombinant (15)N labeled RicC3, a 2S albumin protein from the seeds of castor bean (Ricinus communis), has been determined by NMR methods. The computed structures were based on 1564 upper limit distance constraints derived from NOE cross-correlation intensities measured in the 2D-NOESY and 3D-HSQC-NOESY experiments, 70 phi torsion angle constraints obtained from (3)J(HNH)(alpha) couplings measured in the HNHA experiment, and 30 psi torsion angle constraints derived from (3)J(H)(alpha)(Ni+1) couplings measured in the HNHB experiment. The computed structures showed a RMSD radius of 0.64 A for the structural core. The resulting structure consists of five amphipatic helices arranged in a right-handed super helix, a folding motif first observed in nonspecific lipid transfer proteins. Different than the latter, RicC3 does have not an internal cavity, a fact that can be related to the exchange in the pairing of disulfide bridges in the segment.CXC. Previous attempts to determine high resolution structures of a 2S albumin protein by either X-ray crystallography or NMR methods failed because of the heterogeneity of the protein prepared from natural sources. Both 2S albumins and nonspecific lipid transfer proteins belong to the prolamine superfamily, some of whose members are food allergens. The solution structure for recombinant RicC3 determined here is a suitable representative structure for the broad family of seed 2S albumin proteins, which may help to establish meaningful relationships between structure and allergenicity. RicC3 is also the peptidic component of the immunomodulator Inmunoferon, a widely used pharmaceutical product, and its structure is expected to help understand its pharmaceutical activity.     

Suppression of telomere-binding protein gene expression represses seed and fruit development in tomato

Tomato (Solanum lycopersicum L.) plants were transformed with an antisense construct of a cDNA encoding tomato telomere-binding protein (LeTBP1) to describe the role of a telomere-binding protein at the whole plant level. Fruit size decreased corresponding to the degree of suppression of LeTBP1 expression. This inhibition of fruit development was likely due to a decrease in the number of seeds in the LeTBP1 antisense plants. Pollen fertility and pollen germination rate decreased in accordance with the degree of suppression of LeTBP1 expression. Ovule viability was also reduced in the LeTBP1 antisense plants. Although plant height was somewhat reduced in the antisense plants compared to the control plants, the number and weight of leaves were unaffected by LeTBP1 suppression. The number and morphology of flowers were also normal in the antisense plants. These indicate that reduced fertility in the antisense plants is not an indirect effect of altered vegetative growth. LeTBP1 expression was sensitive to temperature stress in wild-type plants. We conclude that LeTBP1 plays a critical role in seed and fruit development rather than vegetative growth and flower formation.     

Characterization of seed storage protein patterns of Heliotropium digynum

Heliotropium digynum, is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8) software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir’iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.     

Unexpectedly higher expression levels of a chimeric 2S albumin seed protein transgene from a tandem array construct

In wild-type Arabidopsis seeds the 2S albumin seed protein gene family members are differentially expressed. In this work it is shown that as predicted by the wild type situation, the at2S2 promoter is much more effective than that of the at2S1 gene in the expression of a transgene. However, unexpectedly high expression levels were obtained using a construct in which the transgene was present as a tandem duplication in the T-DNA. Neither in this case nor in homozygous plants with either construct was epigenetic silencing observed. While transgene mRNA levels were of the same order of magnitude as the endogenous at 2S2 gene, protein levels were much lower.     

Deletion analysis of a 2S seed storage protein promoter of Brassica napus in transgenic tobacco

The promoter and upstream region of the Brassica napus 2S storage protein napA gene were studied to identify cis-acting sequences involved in developmental seed-specific expression. Fragments generated by successive deletions of the 5 control region of the napA gene were fused to the reporter gene -glucuronidase (GUS). These constructs were used to transform tobacco leaf discs. Analyses of GUS activities in mature seeds from the transformed plants indicated that there were both negatively and positively acting sequences in the napin gene promoter. Deletion of sequences between –1101 and –309 resulted in increased GUS activity. In contrast, deletion of sequences between –309 and –211 decreased the expression. The minimum sequence required for seed-specific expression was a 196 bp fragment between –152 and +44. Further 5 deletion of the fragment to –126 abolished this activity. Sequence comparison showed that a G box-like sequence and two sequence motifs conserved between 2S storage protein genes are located between –148 to –120. Histochemical and fluorometric analysis of tobacco seeds showed that the spatial and developmental expression pattern was retained in the deletion fragments down to –152. However, the expression in tobacco seeds differed from the spatial and temporal expression in B. napus. In tobacco, the napA promoter directed GUS activity early in the endosperm before any visible activity could be seen in the heart-shaped embryo. Later, during the transition from heart to torpedo stages, the main expression of GUS was localized to the embryo. No significant GUS activity was found in either root or leaf.     

A Mycoplasma hyorhinis protein with sequence similarities to nucleotide-binding enzymes.

We have determined the nucleotide (nt) and deduced amino acid (aa) sequence of a unique 115-kDa Mycoplasma hyorhinis protein (P115) with an N-terminal region containing a highly conserved consensus sequence characteristics of nt-binding domains of several ATPase and GTPase enzymes. However, P115 lacked additional conserved features characteristic of some classes of nt-binding proteins. Based on the hydropathy profile of the deduced aa sequence, the absence of a leader peptide, its exclusive partitioning into the hydrophilic phase during Triton X-114 phase fractionation of M. hyorhinis, and immunofluorescence analysis indicating no surface-exposed domains, it was concluded that P115 is a cytoplasmic protein lacking intrinsic membrane interaction. M. hyorhinis P115 appears to be a species-specific protein, since it was not detected in any other mycoplasmal or bacterial species examined with specific antibody or genomic probes. Since genetic systems for direct mutational analysis are currently unavailable in this organism, sequence analysis provides critical information in establishing the possible function of this protein. Moreover, the nt sequence encoding P115 reported here supports a previously proposed model, based on synthesis of P115-related proteins in Escherichia coli, suggesting that multiple polypeptide products can be generated from mycoplasma genes by promiscuous translation initiation in this heterologous expression system.     

Homology of placental protein 11 and pea seed albumin 2 with vitronectin.

Vitronectin (complement S-protein), a plasma and tissue glycoprotein of 75 kDa, shares the amino-terminal somatomedin B domain with the membrane glycoprotein PC1 of plasma cells and several hemopexin-type repeats with hemopexin and the non-catalytic carboxy-terminal domain of collagenases. It serves as a ligand for certain integrin receptors, binds to distinct members of the serpin family and inhibits the pore-forming cytolytic reaction of the terminal complement pathway. Computer-assisted data base searches revealed the presence of a single somatomedin B domain in the recently cloned placental protein 11, and four hemopexin-type repeats in the cytosolic plant protein PA2, the major albumin of pea seeds, whose function is unknown. Our finding shows that hemopexin-type repeats are present in extracellular as well as in cytosolic proteins and most likely originated before the divergence of the animal and plant kindoms.     

The role of endogenous gibberellin in seed and fruit development of tomato: Studies with a gibberellin-deficient mutant

The role of endogenous gibberellin (GA) in seed and fruit development was studied with the use of the GA-deficient ga-1 mutant of tomato ( Lycopersicon esculentum Mill. cv. Moneymaker). Flowers of the ga-1 mutant were abnormal and sterile, but parthenocarpic fruit development was observed occasionally on the dwarf plants. A single application of GA₄₊₇ restored the fertility of the mutant flowers and resulted in seed set. Development of GA-producing and GA-deficient seeds in GA-deficient fruits was compared by pollination of ga-1/ga-1 flowers with wild-type or ga-1 pollen, respectively. In ga-1/ga-1 seed dehydration started about 1 week earlier than in Ga-1/ga-1 seeds. Ultimate fresh and dry weights of mature Ga-1/ga-1 seeds were higher than those of ga-1/ga-1 seeds and showed negative correlations with the total number of seeds per fruit. Total content and composition of seed proteins were not influenced by the GA-deficiency. Germination of the mature seeds depended on embryonal GA synthesis and was not influenced by maternal GA production. Final fresh weight of the ga-1/ga-1 fruits was positively correlated with the number of seeds per fruit. In these fruits, the minimum number of seeds for growth above the parthenocarpic level was about 10 or 35 in the presence of Ga-1/ga-1 or ga-1/ga-1 seeds, respectively. Fruits containing GA-producing seeds reached a higher fresh weight than those containing GA-deficient seeds, and their ripening was delayed by one week. It is concluded that gibberellin is indispensable for the development of fertile flowers and for seed germination, but only promoting in later stages of fruit and seed development.