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.     

Identification of defence proteins from the seed exudates of Cicer arietinum L. and its effect on the growth of Fusarium oxysporum f.sp. ciceri

Germinating seeds tend to release a variety of proteins into their surrounding surfaces; some of which have an inhibitory action against plant pathogens. The aim of this study was to investigate and identify defence proteins present in the exudates from water-imbibed and chitosan-imbibed (0.1% w/v) seeds of chickpea (Cicer arietinum L). Chickpea seeds imbibed in chitosan released a higher amount of proteins in the exudate when compared to the seeds imbibed in water. The obtained exudates were analysed in regard to specific protein activities by enzymatic assays and SDS-PAGE analysis. Results showed that the exude obtained from chickpea seeds imbibed in chitosan solution exhibited a new isoform of chitinase, chitosanase and protease inhibitors. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus, Fusarium oxysporum f.sp. ciceri. Our results suggest that seed exudates protect seeds during their germination from soil pathogens.     

Protein Patterns, Characterized by Computer Image Analysis, of Lentil Embryo Axes Germinating Under Salt Stress

Following 16, 40 and 64 h exposure to 0.33 M NaCl given after 8 h water imbibition, lentil seeds showed a gradual decrease of germination upon their transfer to water. These salt related changes were accompanied by modifications in the protein patterns of embryo axes as revealed by two-dimensional electrophoresis separation and by the computer image analysis of protein spots. In comparison with 8 h water imbibed seeds, prominent proteins comprised between the 5.1 – 7.6 pH isoelectric point in the first dimension and 75 – 50 kDa molecular mass in the second dimension showed a significant increase in their abundance as salt exposure increased. On transfer to water to complete germination, the content of many of these proteins decreased at 24h in 2 – 3 cm length embryo axes in comparison with the corresponding embryo axes of seeds continuously imbibed in water for 24 h. Some groups of proteins ranging between 15.5 – 17.3 kDa, already present after 8 h water imbibition, were not detectable after 24 h but were expressed in seeds exposed to NaCl and transferred to water for 24 h. Up- and down-regulated proteins in lentil embryo axes, imbibed under non-lethal salt stress conditions, have been tentatively identified by comparison with the protein map of germinating seeds of the model plant Arabidopsis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97–100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52–56 h and 64–72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.     

Free amino acids in the seed and root exudates in relation to the nitrogen requirements of rhizosphere soil Fusaria

Summary The amounts of amino acids in seed exudates were generally higher than in root exudates of the same plant. The spectra and relative abundance of amino acids in both plants were similar but they were generally more abundant in cowpea exudates than in sorghum. Glutamic acid and alanine were the most abundant amino acids in the seed and root exudates of both plants. The proportions of the amino acids in the seed exudates were comparable to that stored in the seeds. Many of the major amino acids identified in the exudates were also found to support thein vitro growth ofFusarium spp. isolated from the rhizosphere and rhizoplane. This suggests that the amino acids exuded might contribute signficantly to Fusaria nutrition and its consequent predominance around the root. The significance of this pathogenesis is also discussed.     

Comparative Proteomics Reveals the Mechanisms Underlying Variations in Seed Vigor Based on Maize (Zea mays L.) Ear Positions

Seed vigor is influenced by seed position in plant. However, current understanding of its underlying mechanism is limited. In this study, we used isobaric tags for relative and absolute quantitation technique to study the comparative proteomes between middle seeds (with higher vigor) and top seeds of maize (Zea mays L.) ears at 0 h, 24 h, and 48 h of imbibition. A total of 159 differentially accumulated proteins were identified. Among these, the largest number of proteins was from the functional categories of Disease/Defense and Metabolism. Compared with top seeds, most of the differentially accumulated proteins of Protein Synthesis and Energy showed higher accumulation in middle seeds at 0 h and 24 h of imbibition, but lower accumulation at 48 h of imbibition. Seed water absorption activates metabolic processes. The water content of middle seeds was significantly lower than that of top seeds at between 12 h and 30 h of imbibition, but energy production would be higher in the middle seeds at 24 h of imbibition. Meanwhile, tonoplast intrinsic proteins 3.1 and 3.2, which mediate water inflow into protein storage vacuoles, then activating enzymes involved in reserve mobilization, showed higher accumulation in middle seeds at 24 h of imbibition. In addition, our data also showed middle seeds may suffer less fungal damages. Our results contribute to understanding the mechanisms underlying the effects of growth position on seed vigor.

     

Patterns of protein synthesis during the germination of pea axes,and the effects of an interrupting desiccation period

As germination of axes of Pisum sativum L. seeds progressed, profound quantitative and qualitative changes occurred in the patterns of protein synthesis. This was shown by fluorography of gels following two-dimensional polyacrylamide gel electrophoresis separation of [³⁵S]methioninelabelled proteins. The effects of desiccation during germination on these in-vivo protein-synthesis patterns were followed. Desiccation differentially affected the synthesis of proteins. Usually, however, upon rehydration following desiccation the types of proteins being synthesized were recognizable as those synthesized earlier during imbibition of control, once-imbibed axes: seeds imbibed for 8 h, and then dried, did not recommence synthesis of proteins typical of 8-h-imbibed control seeds, but rather of 4-h-imbibed control seeds. Seeds imbibed for 12 h, and then dried and rehydrated, synthesized proteins typical of 4-h-and 8-h-control seeds. Thus drying of germinating pea axes caused the proteinsynthesizing mechanism to revert to producing proteins typical of earlier stages of imbibition. Drying during germination never caused the seed to revert to the metabolic status of the initial mature dry state, however.Abbreviation DR dried and rehydrated     

Changes in concentrations of soluble carbohydrates during germination of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. seeds in relation to ethylene,gibberellin A<Subscript>3</Subscript> and methyl jasmonate

Unimbibed Amaranthus caudatus seeds were found to contain stachyose, raffinose, verbascose, sucrose, galactinol, myo-inositol, glucose and fructose, while no galactose, maltose and maltotriose was detected. During imbibition, seed concentrations of verbascose, stachyose, raffinose, galactinol, myo-inositol (temporary) and fructose (transient) were observed to decrease; concentrations of galactose and maltose remained fairly constant, while those of sucrose, glucose and maltotriose increased, the increase in sucrose concentration was only temporary. Effects of gibberellin A₃ (GA₃) at 3 × 10⁻⁴ M and ethephon at 3 × 10⁻⁴ M alone or in the presence of methyl jasmonate (Me-JA) at 10⁻³ M on concentrations of soluble sugars during germination of A. caudatus seeds were examined. Me-JA was found to inhibit seed germination and fresh weight of the seeds, but did not affect sucrose, myo-inositol, galactose and maltose concentrations during imbibition for up to 20 h. The exogenously applied GA₃ was observed to enhance germination, stachyose breakdown and glucose concentration after 20 h of incubation. Ethephon stimulated seed germination as well as utilisation of stachyose, galactinol (both after 14 and 20 h) and raffinose (after 14 h of incubation). Although the stimulatory effect of either GA₃ or ethephon on seed germination was blocked by Me-JA; these stimulators increased mobilisation of raffinose and stachyose, but only ethephon enhanced both glucose and fructose after 14 and/or 20 h of incubation in the presence of Me-JA. The maltose concentration was increased by both GA₃ and ethephon alone and in the presence of Me-JA. Of the growth regulators studied, ethephon alone and/or in combination with Me-JA significantly increased the concentrations of glucose, fructose, galactose, maltose and maltotriose. The differences in sugar metabolism appear to be linked to ethylene or GA₃ applied simultaneously with Me-JA.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Fruits foraging patterns and seed dispersal effect of frugivorous birds on Hippophae rhamnoides sinensis

Behaviors of 18 species of birds eating fruits of Hippophae rhamnoides spp. sinensis were observed from September 2003 to March 2004. Their foraging patterns were found to be very different and can be divided into five classes: (1) direct swallowing the fruits on crown of the shrubs and sometimes regurgitating seeds soon after; (2) carrying the fruits to their perching sites and swallowing; (3) pecking the fruits from the shrubs to the ground, eating pulp and seeds but leaving pericarp; (4) pecking through the pericarp, eating pulp and leaving pericarp and seeds; (5) pecking through the pericarp on the top of fruits, and only eating seeds. These foraging patterns have different effects on seed dispersal of H. rhamnoides spp. sinensis. The germination experiment of three groups of seeds (seeds from feces, dry fruits and extracted seeds from dry fruits) was carried out. Although ingestion processes of birds had some adverse effects on the seed germination of H. rhamnoides spp. sinensis, the seeds from feces still have a relatively higher germination ratio. H. rhamnoides spp. sinensis provides food to a variety of frugivorous birds, and the birds disperse its seeds. Thus, a mutually beneficial relationship between the bird and the seed is formed. __________ Translated from Chinese Journal of Ecology, 2005, 24(6): 635–638 [译自: 生态学杂志, 2005, 24(6): 635–638]     

Metabolic changes in tomato fruits and seeds after viral, bacterial and fungal infections

The metabolic changes in tomato fruits and seeds separately infected with cucumber mosaic virus, Pseudomonas syringae pv. tomato or Botrytis cinerea were investigated cytochemically. The changes of peroxidase (E.C. 1.11.1.7) and β-glucosidase (E.C. 3.2.1.21) were investigated biochemically as well. Tomato fruits were involved in the study because of their high economic value. Tomato seeds were investigated since they have been used most extensively as a model system for studying the physiology and biochemistry of seed development. The diseases caused by the pathogens under study are of special importance for yield reduction in tomato. The three pathogens provoked local changes in the activities of enzymes under study that affect the infected pericarp tissues and neighboring seeds. It was established non-specific and specific responses. The non-specific responses of invaded tissues were expressed as a local enhancement of peroxidase activity in both pericarp tissues and seeds as well as a decrease in activities of: i. enzymes taking part in aerobic and anaerobic respiration, ii. hydrolases esterase and acid phosphatase involved in the basic metabolism as well as an enhancement of their activities in neighboring tissues. Furthermore, it was observed an enhancement of α-galactosidase activity in infected area was observed. The specific responses depending on the type of the pathogen consisted in an enhancement of glucose-6-phosphate dehydrogenase activity by virus infection and an increase of β-glucosidase activity by fungal invasion.     

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.     

D-Amino-acid-stimulated ethylene production in seed tissues

Ethylene production by axial and cotyledonary tissues excised from Xanthium pennsylvanicum Wallr. seeds was markedly (up to 5-fold) stimulated by the D-isomers of phenylalanine, valine, leucine, threonine, methionine and eithionine while the L-isomers caused no such effect. Responsiveness of these seed tissues to D-methionine appeared soon after the beginning of imbibition, reached a maximum after 6–12 and 12–24 h for the axial and cotyledonary tissues, respectively, and then decreased sharply. D-Phenylalanine and D-methionine also stimulated ethylene production in seed tissues of X. canadense Mill. and in cotyledonary segments from seeds of Helianthus annuus L., Cucurbita moschata Duch. and Vigna radiata (L.) Wilczek. The endogeneous ethylene production and the D-amino-acid-stimulated ethylene production by the seed segments was strongly inhibited by aminoethoxyvinyl glycine, a potent inhibitor of ethylene synthesis from L-methionine.     

Heterologous expression of IAP1, a seed protein from bean modified by indole-3-acetic acid,in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The seed protein IAP1 from bean (PvIAP1; Phaseolus vulgaris L.) that is modified by the phytohormone indole-3-acetic acid (IAA) was heterologously expressed in the two reference plant species Arabidopsis thaliana and Medicago truncatula. For the transformation of Medicago we devised a novel protocol using seedling infiltration. When PvIAP1 was overexpressed under the control of the constitutive 35SCaMV promoter in Arabidopsis, the plants showed signs of earlier bolting and enhanced branching. Expression of a fusion protein of PvIAP1 with both a green fluorescence protein (GFP) as reporter and 6× histidine (His) tag under the control of the native bean IAP1 promoter resulted in the accumulation of the protein in both plant species exclusively in seeds as shown by immunoblotting and by fluorescence microscopy. During seed development, PvIAP1 was first expressed in the vascular bundle of Arabidopsis, whereas in later stages GFP fluorescence was visible essentially in all tissues of the seed. Fluorescence decreased rapidly after imbibition in the seeds for both Arabidopsis and Medicago, although the fluorescence persisted longer in Arabidopsis. GFP fluorescence was distributed evenly between an organelle fraction, the microsomal membrane fraction, and the cytosol. This was also confirmed by immunoblot analysis. Clusters of higher GFP fluorescence were observed by confocal microscopy. Although PvIAP1 protein accumulated in seeds of both Arabidopsis and Medicago, neither species post-translationally modified the protein with an indoleacyl moiety as shown by quantitative GC–MS analysis after alkaline hydrolysis. These results indicate an apparent specificity for IAA attachment in different plant species. Alexander Walz and Claudia Seidel contributed equally to the paper.     

Striga seed-germination activity of root exudates and compounds present in stems of Striga host and nonhost (trap crop) plants is reduced due to root colonization by arbuscular mycorrhizal fungi

Root colonization by arbuscular mycorrhizal (AM) fungi reduces stimulation of seed germination of the plant parasite Striga (Orobanchaceae). This reduction can affect not only host plants for Striga, resulting in a lower parasite incidence, but also false hosts or trap crops, which induce suicidal Striga seed germination, thereby diminishing their effectiveness. In order to better understand these AM-induced effects, we tested the influence of root colonization by different AM fungi on the seed-germination activity of root exudates of the Striga hermonthica nonhost plants cowpea and cotton on S. hermonthica. We also tested the effect of AM fungi on the seed-germination activity of the Striga gesnerioides host plant cowpea on S. gesnerioides. Moreover, we studied whether mycorrhization affects the transport of seed-germination activity to above-ground plant parts. Mycorrhization not only resulted in a lower seed germination of S. gesnerioides in the presence of root exudates of the S. gesnerioides host cowpea but also seed germination of S. hermonthica was also lower in the presence of root exudates of the S. hermonthica nonhosts cowpea and cotton. Downregulation of the Striga seed-germination activity occurs not only in root exudates upon root colonization by different AM fungi but also in the compounds produced by stems. The lowered seed-germination activity does not appear to depend on the presence of seed germination inhibitors in the root exudates of mycorrhizal plants. The implication for Striga control in the field is discussed.     

Effects of harvester ants on seed availability and dispersal of Lavandula stoechas subsp. pedunculata in a Mediterranean grassland-scrubland mosaic

The role of harvester ants in Mediterranean grassland and scrubland has mostly focused on seed consumption. However, recent studies have reported their role as accidental dispersal agents of some of the collected seeds via refuse piles. The objective of this study is to examine the effect of the ant Messor barbarus on seed availability and dispersal of one of its major diet components, Lavandula stoechas subsp. pedunculata, in scrubland, grassland and the ecotones between them. After confirming and quantifying the Lavandula contribution to M. barbarus diet, we described the spatial and temporal patterns of pre- and post-dispersal seed predation, seed content and seedling occurrence in the refuse piles. Our results show that: (1) Lavandula propagules constitute a high proportion of the prey items collected by M. barbarus, with particularly intense collection activity in mid-summer, spring and autumn, in decreasing order. (2) Pre-dispersal predation rate was significantly higher in the ecotone than in the scrubland (76% and 13.5% of total seed production lost respectively). (3) Season and propagule type (seed vs. fruit) were the most significant variables explaining the post-dispersal predation probability, which approached 100% of seeds after 48 h in mid-summer. (4) Viable Lavandula seeds were found in refuse piles at densities of 0.06–0.2 per g of refuse pile material, or 58.8–207.2 per refuse pile. On the one hand, these results indicate that the ecotones are most affected by M. barbarus pre-dispersal consumption, which may locally limit Lavandula colonisation. On the other hand, the small proportion of consumed seeds that is dispersed to refuse piles may be relevant at the population level, as this dispersal implies arrival at potentially favourable sites for establishment.     

Seed weight increases with altitude in the Swiss Alps between related species but not among populations of individual species

Seed weight is a crucial plant life history trait, determining establishment success and dispersal ability. Especially in stressful environments, larger seeds may be selected at the expense of seed number, because larger seeds have a better chance of giving rise to an established offspring. We tested the hypotheses that between related species-pairs and among populations of single species a similar trend for increasing seed weight with increasing altitude should be present. Firstly, we measured seed weights from 29 species-pairs, with one species occurring in lowland areas and a congeneric species from high altitudes. Seeds of the alpine species were 28±8% larger than seeds from lowland species (P<0.01). Compared to the related lowland species, 55% of the alpine species had heavier seeds, 3% (one species) had lighter, and 41% had seeds of approximately equal weight. Secondly, we compared seed weights among populations of four species from different habitats and with different life histories. Seeds from between 11 and 34 populations per species were sampled along altitudinal gradients of 800–1,500 m (ca. 800 m in Scabiosa lucida, ca. 1,000 m in Saxifraga oppositifolia, ca. 1,000 m in Epilobium fleischeri, and ca. 1,500 m in Carex flacca). In all the four species, we found no indication for heavier seeds at higher altitudes. Our results indicate a selection pressure for species with heavier seeds at higher altitude, but the trend does not seem to operate across all cases. Phylogenetic constraints may limit the correlation among altitude and seed weight, operating particularly against selection for larger seed size, the closer populations and species are related to each other.     

Proteins and polyamines during dormancy breaking of European beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) seeds

The studies were carried out on Fagus sylvatica seeds during stratification and their germination. After imbibition beechnuts were subjected to cold (3 °C — temperature which breaks dormancy) or warm (15 °C — temperature unable to break dormancy) stratification and alternatively were treated with polyamine synthesis inhibitors: canavanine and DFMO (difluoromethylornithine). After cold stratification in embryo axes we found (using 2-D electrophoresis) about 150 new proteins absent in dry seeds. Exogenous spermidine increased the protein synthesis, percent of germinated seeds and accelerated breaking of dormancy. In contrast, canavanine and DFMO decreased dynamic of protein synthesis, quantity of proteins probably synthesised de novo, and percent of germinated seeds. The maximum of polyamine content in embryo axes during cold stratification preceded such the maximum during warm stratification. Irrespective of the influence of PAs and inhibitors of PA synthesis, the comparison of electrophoregrams and autoradiograms showed that different groups synthesised de novo appeared after different periods of cold stratification. Probably the part of this protein is associated with Fagus sylvatica seeds dormancy breaking.