Defense Proteins from Vigna Unguiculata Seed Exudates: Characterization and Inhibitory Activity Against Fusarium Oxysporum

Plants exude a variety of substances through their roots, germinating seeds and aerial parts. Some of these released compounds seem to have an inhibitory effect against pathogens. The aim of this work was to investigate and identify antifungal proteins present in exudates from imbibed cowpea seeds (Vigna unguiculata (L.) Walp). The obtained exudation was analyzed in regard to specific protein activities by enzymatic or immunological assays for plant defense proteins, from 4 h to 48 h of seed imbibition. Our results show that cowpea seeds exudates present several defense related proteins characterized as β-1,3-glucanases, cystatins, vicilins and lipid transfer proteins (LTPs), as well as a storage vacuole membrane α-TIP protein, since the very first hours of imbibition. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus Fusarium oxysporum f. sp. phaseoli. Our results suggest that seed exudates should promote seed protection from soil pathogens.     

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.     

Antimicrobial proteins from cowpea root exudates: inhibitory activity against Fusarium oxysporum and purification of a chitinase-like protein

Plants exude a variety of substances through their external surfaces and from germinating seeds, some of which have an inhibitory action against plant pathogens. The aim of this study was the investigation and characterization of defense proteins present in exudates from roots of cowpea seedlings (Vigna unguiculata (L.) Walp.). Root exudates were collected from seedlings that were grown hydroponically in three different media, including, 100 mM sodium acetate buffer pH 4.5, water pH 6.0 and 100 mM sodium phosphate buffer pH 7.5. The proteins from these exudates were analyzed by SDS–PAGE and SDS–Tricine–PAGE and the presence of antimicrobial proteins in the exudates was investigated by immunological and enzymatic assays. Results showed that roots from cowpea seedlings contained -1,3-Glucanases, chitinases and lipid transfer proteins (LTPs), all of which may potentially function as plant defense proteins. Immunolocalization of one of these proteins, chitinase, revealed its presence in the xylem cell wall vessel elements. These exudates also demonstrated an inhibitory effect on the growth of the fungus, Fusarium oxysporum, in vitro. The results suggest that plant roots may exude a variety of proteins that may function to repress the growth of root pathogenic fungi.     

Proteome analysis of embryogenic cell suspensions of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>)

Using a combination of two-dimensional gel electrophoresis protein mapping and mass spectrometry analysis, we have established proteome reference maps of embryogenic cell suspensions of cowpea (Vigna unguiculata). The cell suspensions were generated from young primary leaves and contained basically pro-embryogenic masses, which enabled us to dissect their proteome composition while eliminating the complexity of too many cell types. Over 550 proteins could reproducibly be resolved over a pI range of 3–10. A total of 128 of the most abundant protein spots were excised, digested in-gel with trypsin and analyzed by tandem mass spectrometry. This enabled the identification of 67 protein spots. Two of the most abundant proteins were identified as a chitinase and as a ribonuclease belonging to the family of PR-4 and PR-10 proteins, respectively. The expression of the respective genes was confirmed by RT-PCR and the pattern of deposition of the PR-10 protein in cell suspensions as well as in developing cowpea seeds, roots, shoots and flowers were determined by Western blot experiments, using synthetic antibodies raised against a 14-amino acid synthetic peptide located close to the C-terminal region of the PR-10 protein.     

Identification and pathogenic characterization of endophytic <Emphasis Type="Italic">Fusarium</Emphasis> species from cowpea seeds

Isolates of Fusarium were obtained and identified from seeds of cowpea, Vigna unguiculata (L.) Walp., by means of blotter tests and slide cultures. Species were differentiated according to the morphology of the macroconidia, microconidia and their arrangement in chains or false heads, the size and type of conidiophore, and the presence or absence of chlamydospores. The species were identified as F. semitectum, F. equiseti, F. oxysporum, F. solani, F. anthophilum, F. sporotrichioides, F. moniliforme, and Fusarium sp. Among the species, F. semitectum was the most frequently detected. None of these species were pathogenic when inoculated in susceptible cowpea cultivar (BR 17- Gurgueia). But, an isolate of F. oxysporum f. sp. tracheiphilum used as a standard of comparison for pathogenicity (control) induced symptoms of yellowing, vascular wilting, and death of a susceptible cowpea cultivar under the same environmental conditions.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

Seed size variation in the palm Euterpe edulis and the effects of seed predators on germination and seedling survival

Intraspecific variation in seed size is common in wild plant populations and has important consequences for the reproductive success of individual plants. Multiple, often conflicting evolutionary forces mediated by biotic as well as abiotic agents may maintain such a variation. In this paper we assessed seed size variation in a population of the threatened, commercially important palm Euterpe edulis in southeast Brazil. We investigated (i) how this variation affects the probability of attack by vertebrate and invertebrate post-dispersal seed predators, and (ii) if seed size influences the outcome of seeds damaged by beetles in terms of seed germination and early survival of seedlings. Euterpe edulis seeds varied in diameter from 8.3 to 14.1 mm. Neither insects nor rodents selected the seeds they preyed upon based on seed size. Seed germination and total, shoot and root biomasses of one-year seedlings were significantly and positively affected by seed size. Root biomass and seedling survival were negatively affected by seed damage caused by a scolytid beetle (Coccotrypes palmarum) whose adults bore into seeds to consume part of the endosperm, but do not oviposit on them. Seed size had a marginally significant effect on seedling survival. Therefore, if any advantage is accrued by E. edulis individuals producing large seeds, this is because of greater seed germination success and seedling vigor. If this is so, even a relatively narrow range of variation in seed size as observed in the E. edulis population studied may translate into differential success of individual plants.     

Unusual sequence and characteristics of a chick-pea seed protein which is regulated by abscisic acid and is similar to late-embryogenesis-abundant proteins

The cDNA clone GAB-9 was selected from a cDNA gene library constructed from the mRNA of embryonic axes of chick-pea (Cicer arietinum L.) seeds imbibed for 12 h in the presence of abscisic acid. The sequence of this cDNA has an open reading frame of 546 nucleotides that code for 182 amino acids. The polypeptide encoded by the corresponding mRNA is of approx. 20.5 kDa, is basic, and has a broad hydrophobic central region flanked by two hydrophilic regions. The unusual characteristics of this protein, which is similar to late-embryogenesis-abundant proteins, and its possible function are discussed.Abbreviations ABA abscisic acid - HSP heat-shock proteins - LEA late embryogenesis abundant The nucleotide sequence data reported appear in the EMBL under the accession number X79680.We thank Dr J.L. Revuelta for helpful discussion. This work was supported by grants from Direction General de Investigation Científica y Técnica, Spain (PB90-0536) and Junta de Castilla y León (SA-33/11/92).     

Resistance function of rice lipid transfer protein LTP110

Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures.     

Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization

Three cDNA clones encoding lipid transfer proteins (LTPs) were isolated by applying the rapid amplification of cDNA ends (RACE) protocol to imbibed seeds and germinating seedlings of Brassica napus. The deduced amino-acid sequences show a great degree of homology and they exhibit the common features shared by all LTPs. Their expression pattern indicates a strong developmental, hormonal, and environmental regulation. They are expressed only in cotyledons and hypocotyls of germinating seedlings and their levels of expression increase upon treatment with cis-abscisic acid and NaCl. Their distribution in the cotyledons of young seedlings is suggestive of a role related to the mobilization of lipid reserves.     

Lipid transfer protein genes specifically expressed in barley leaves and coleoptiles

Genes/cDNAs encoding so-called lipid-transfer proteins (LTPs) have been isolated from a variety of tissues from different plants, but the in-vivo function of the LTP proteins is not yet known. In barley (Hordeum vulgare L.), the LTP1 gene (encoding a probable amylase/ protease inhibitor, Mundy and Rogers 1986, Planta 169, 51–63) is active in aleurone tissue, and in this paper two LTP-encoding cDNAs isolated from green leaves are described. The encoded proteins start with signal sequences, they are 75% homologous to each other, 60–63% homologous to rice aleurone LTP and maize seed/ coleoptile LTP, but only 48% homologous to barley aleurone LTP. Northern hybridization experiments established that the two seedling-specific genes are both highly expressed in leaves and coleoptiles whereas the LTP1 gene is inactive in seedlings. No LTP gene expression was detected in roots using either seedling or aleurone cDNA clones as probes. Tissue-print hybridization indicates that the LTP genes are first expressed in young epidermal cells in leaves and coleoptiles, and subsequently expressed in the vascular strands. Genomic Southern analysis indicates that the barley LTP gene family has four to six members.Abbreviation LTP lipid transfer protein I thank Dr. J. Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark for the PAPI cDNA clone and R. Barkardottir, Department of Molceular Biology, University of Aarhus, Denmark for providing RNA for some of the Northern analyses. I also thank I. Bjørndal and L. Kjeldbjerg for excellent technical assistance. This work was supported by the The Danish Biotechnology Programme.     

Depth distribution and composition of seed banks under different tree layers in a managed temperate forest ecosystem

In the present work we examined the composition and distribution across three soil layers of the buried soil seed bank under three different overstory types (Fagus sylvatica, Quercus robur, Pinus sylvestris) and in logging areas in a 4383-ha forest in central Belgium. The objectives were: (1) to investigate whether species composition and species richness of soil seed banks are affected by different forest stands; (2) to examine how abundant are habitat-specific forest species in seed banks under different planted tree layers. The study was carried out in stands which are replicated, managed in the same way (even-aged high forest), and growing on the same soil type with the same land-use history. In the investigated area, the seed bank did show significant differences under oak, beech, pine and in logging areas, respectively in terms of size, composition and depth occurrence. All species and layers taken together, the seed bank size ranked as follows: oakwood > beechwood > logging area > pinewood. The same pattern was found for forest species. Seed numbers of Betula pendula, Calluna vulgaris, Dryopteris dilatata and Rubus fruticosus were significantly higher under the beech canopy. Carex remota, Impatiens parviflora and Lotus sp. showed a significantly denser seed bank in logging areas, while Digitalis purpurea seeds were significantly more abundant in soils under the oak canopy. The fact that the seed bank of an originally homogeneous forest varies under different planted stands highlights that a long period of canopy conversion can affect the composition and depth of buried seeds.     

Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development

We have analyzed the nucleotide sequence and accumulation of an mRNA which is prevalent in seeds of Brassica napus L. During normal development, the mRNA begins to accumulate during late embryogeny, is stored in dry seeds, and becomes undetectable in seedlings within 24 hours after imbibition. Moreover, abscisic acid treatment of embryos precociously induces or enhances accumulation of the mRNA. Nucleotide sequencing studies show that the deduced 30 kDa polypeptide has an unusual primary structure; the polypeptide possesses direct amino acid sequence repeats and is virtually entirely hydrophilic with the exception of a hydrophobic carboxyl-terminal region. Based upon the expression pattern and predicted polypeptide sequence, we conclude that the mRNA is encoded by a late embryogenesis-abundant (Lea) gene in B. napus.Abbreviations ABA abscisic acid - bp base pairs - DAF days after flowering - HAI hours after the start of imbibition - kb kilobase (pairs)     

Use of micro-CAT scans to understand cowpea seed resistance to Callosobruchus maculatus

The cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchini), is a worldwide pest of stored cowpea grain [ Vigna unguiculata (L.) Walp. (Leguminosae)], causing tens of millions of dollars' worth of damage annually. One method of managing this pest involves planting cultivars whose seeds are resistant to bruchids. Despite extensive research, we still do not know the mechanism by which TVu 2027, the original resistant cowpea line, resists C. maculatus . Using micro-CAT (computerized axial tomography) scan imaging, we visualized the feeding patterns of bruchids living and growing within cowpea seeds. We present evidence that an interior zone in TVu 2027 seeds is responsible for the high mortality and developmental delays experienced by avirulent larval bruchids. We observed that both virulent as well as avirulent bruchid strains have different feeding patterns in TVu 2027 seeds compared to susceptible seeds. It appears that the resistance factor is most concentrated in a zone in each cotyledon adjacent to the air space separating the two seed halves.