Effect of Canavanine from Alfalfa Seeds on the Population Biology of Bacillus cereus

Bacillus cereus UW85 suppresses diseases of alfalfa seedlings, although alfalfa seed exudate inhibits the growth of UW85 in culture (J. L. Milner, S. J. Raffel, B. J. Lethbridge, and J. Handelsman, Appl. Microbiol. Biotechnol. 43:685–691, 1995). In this study, we determined the chemical basis for and biological role of the inhibitory activity. All of the alfalfa germ plasm tested included seeds that released inhibitory material. We purified the inhibitory material from one alfalfa cultivar and identified it as canavanine, which was present in the cultivar Iroquois seed exudate at a concentration of 2 mg/g of seeds. Multiple lines of evidence suggested that canavanine activity accounted for all of the inhibitory activity. Both canavanine and seed exudate inhibited the growth of UW85 on minimal medium; growth inhibition by either canavanine or seed exudate was prevented by arginine, histidine, or lysine; and canavanine and crude seed exudate had the same spectrum of activity against B. cereus, Bacillus thuringiensis, and Vibrio cholerae. The B. cereus UW85 populations surrounding canavanine-exuding seeds were up to 100-fold smaller than the populations surrounding non-canavanine-exuding seeds, but canavanine did not affect the growth of UW85 on seed surfaces. The spermosphere populations of canavanine-resistant mutants of UW85 were larger than the spermosphere populations of UW85, but the mutants and UW85 were similar in spermoplane colonization. These results indicate that canavanine exuded from alfalfa seeds affects the population biology of B. cereus.     

Canavanine distribution in jackbean fruit during fruit growth

Summary Canavanine is an arginine analogue found in the seeds of many common legumes and is known to inhibit protein synthesis and growth in a number of organisms. Yet canavanine may comprise as much as 4% of the seed dry weight of the jackbean (Canavalia ensiformis).Canavanine is accumulated during earlier development in the pod, but disappears upon ripening. A corresponding increase in seed canavanine of about the same magnitude as the loss in the pod takes place during this latter time, but there is a subsequent significant increase of canavanine content of the seed after all detectable canavanine has disappeared from the pod. The first of these changes suggests synthesis of canavanine in the pod and transport into the seeds while the second one indicates a synthesis of canavanine in the seeds themselves, or possibly in the leaf or pod with rapid translocation to the seed.Canavanine was found to be at its highest concentration in the seed coats and pods when they were growing most rapidly and to gradually decline afterwards; however, the canavanine concentration of the seeds was found to be constant throughout fruit development.The pattern of canavanine mobilization in jackbean fruits was quite similar to the known pattern of total nitrogen mobilization typical of other leguminous fruits. This is consistent with a role as a nitrogen transport and storage compound.University of Tennessee, Department of Botany, Contribution N. Ser. No. 279.     

The occurrence of canavanine in seeds of the tribe robinieae

Fifty-five species representing 14 genera of Robinieae are reported to accumulate canavanine in seed. This represents approximately one-third the number of species and two-thirds the number of genera in the tribe and indicate Robinieae to be the only tribe with a predominantly New World distribution to accumulate canavanine. The only member of Robinieaea found not to accumulate canavanine is the genus Diphysa. The lack of canavanine in seed of this genus lends systematic weight to characters shared in common by Diphysa with the tribe Aeschynomeneae, and a re-evaluation of the tribal position of Diphysa may be in order.     

The Interrelationship of Canavanine and Urease in Seeds of the Lotoideae

Seeds of 29 species of canavanine-synthesizing legumes wereassayed for their urease and canavanine production. All of theexamined species possess detectable urease activity. In general,the leguininous seeds richest in urease also had the most canavanine. The urease content of the jack bean seed, Canavalia ensiformis(L.) DC., is formidable and disproportionally greater than thequantity of stored canavanine. The massive urease content ofthe seed cannot be rationalized by the magnitude of the canavaninepool. Analysis of eight species of Mucuna demonstrated that canavanineis not stored in the seeds of these plants. Mucuna species donot appear to be unique in having seeds that do not concurrentlyproduce urease and canavanine.     

The influence of canavanine and related compounds on the water balance system of locusts

In vivo increase in haemolymph volume of canavanine-treated locusts substantiates our previous in vitro findings that canavanine inhibits fluid secretion by locust Malpighian tubules. Furthermore when diuretic hormone is applied in vivo after canavanine treatment haemolymph volume is drastically reduced below levels retained in locusts untreated with canavanine. Again this is in accord with canavanine potentiation of semi-isolated Malpighian tubules and enhanced fluid secretion in vitro. The response is specific to canavanine; compounds similar in structure (arginine, argininic acid, citrulline, canaline, ornithine and homoserine) have no effect on the rate of fluid secreted by Malpighian tubules. Only partial competition is obtained with uridine homoserine.     

Effect of Canavanine on Murine Retrovirus Polypeptide Formation

Canavanine is an arginine analog which is widely used to inhibit proteolytic processing of viral polyproteins. Certain results obtained with canavanine have suggested that it may have other effects. Therefore, we examined the effects of canavanine on the cell-free synthesis of murine retrovirus proteins. It was found that the electrophoretic mobility of the major gag-related cell-free product of both Rauscher murine leukemia virus (R-MuLV) and Moloney murine sarcoma virus 124 (Mo-MuSV-124) RNA was dependent on the concentration of canavanine used during translation. As the canavanine concentration was increased up to 4 mM, the apparent size of the major gag-related polypeptide also increased from 65,000 (R-MuLV RNA) or 63,000 (Mo-MuSV-124 RNA) to approximately 80,000 daltons. Additional increases in the canavanine concentration up to 12 mM did not increase the size of the gag gene product beyond 80,000 daltons. This change in electrophoretic mobility appeared to be due to a substitution of canavanine for arginine residues in the polypeptides, not to a change in their actual size. If amber suppressor tRNA and canavanine were used together during translation of Mo-MuSV-124 RNA and Mo-MuLV RNA, the results were also in agreement with this proposal. Translation experiments done with ovalbumin mRNA and mengovirus 35S RNA indicated that canavanine incorporation caused a shift in the electrophoretic mobility of ovalbumin from 43,000 to 45,000 daltons and caused the appearance of two slightly larger polypeptides in the 155,000- and 115,000- dalton regions of the mengovirus RNA cell-free product.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

The Distribution of Fruit and Seed Toxicity during Development for Eleven Neotropical Trees and Vines in Central Panama

Secondary compounds in fruit mediate interactions with natural enemies and seed dispersers, influencing plant survival and species distributions. The functions of secondary metabolites in plant defenses have been well-studied in green tissues, but not in reproductive structures of plants. In this study, the distribution of toxicity within plants was quantified and its influence on seed survival was determined in Central Panama. To investigate patterns of allocation to chemical defenses and shifts in allocation with fruit development, I quantified variation in toxicity between immature and mature fruit and between the seed and pericarp for eleven species. Toxicity of seed and pericarp was compared to leaf toxicity for five species. Toxicity was measured as reduced hyphal growth of two fungal pathogens, Phoma sp. and Fusarium sp., and reduced survivorship of brine shrimp, Artemia franciscana, across a range of concentrations of crude extract. I used these measures of potential toxicity against generalist natural enemies to examine the effect of fruit toxicity on reductions of fruit development and seed survival by vertebrates, invertebrates, and pathogens measured for seven species in a natural enemy removal experiment. The seed or pericarp of all vertebrate- and wind-dispersed species reduced Artemia survivorship and hyphal growth of Fusarium during the immature and mature stages. Only mature fruit of two vertebrate-dispersed species reduced hyphal growth of Phoma. Predispersal seed survival increased with toxicity of immature fruit to Artemia during germination and decreased with toxicity to fungi during fruit development. This study suggests that fruit toxicity against generalist natural enemies may be common in Central Panama. These results support the hypothesis that secondary metabolites in fruit have adaptive value and are important in the evolution of fruit-frugivore interactions.     

The biosynthesis of canavanine from 14CO2 and its asymmetric labeling in isolated pericarp,tissue of Canavalia ensiformis

Summary Pericarp disks from the fruit of the jackbean (Canavalia ensiformis) when exposed to ¹⁴CO₂ for 2 days carried out photosynthesis and the canavanine extracted from the tissue was labeled with the radioisotope. When beef liver arginase was allowed to react with this canavanine the products were homoserine, canaline, urea and an unknown compound. The activity ratio of C₄:C₁ compounds was close to 2:1. No label could be detected in the canavanine from leaves exposed in the same way to ¹⁴CO₂ and it was concluded that normally canavanine synthesis occurs in the pericarp chlorenchyma.University of Tennessee, Department of Botany, Contribution N. Ser. No. 361.     

Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiformia (L.) DC: I. Canavanine Utilization in the Developing Plant

An ontogenetic study of the canavanine and soluble protein pools in the developing jack bean plant, Canavalia ensiformis (L.) DC., was conducted. Evidence was presented which clearly established the conversion of canavanine to canaline and urea as the principal pathway of canavanine utilization. The catabolic reactions of certain bacteria involving the formation of guanidine or hydroxyguanidine from canavanine are not operative in the cotyledons of jack bean. Evidence was obtained which indicates that a second, minor reaction is functioning in canavanine degradation.     

Endocarpic Microorganisms of Two Types of Windrow-Dried Peanut Fruit (Arachis hypogaea L.)

The endocarpic microorganisms of peanut fruit dried in either a random windrow (plants left as they fell from the digger) or an inverted windrow (plants inverted to expose fruit to sunlight) were different from that of freshly dug fruit. Chaetomium, Penicillium, Trichoderma, Rhizoctonia, and Fusarium were the dominant fungi found associated with shells (pericarp) of freshly dug fruit. The dominant fungi of shells of windrowed fruit included Chaetomium, Rhizoctonia, Fusarium, Sclerotium, and Alternaria. Seeds of freshly dug fruit were dominated by Penicillium and Aspergillus. The only dominant species in seed of windrowed fruit was Penicillium. Microorganisms were isolated from shells and seed of freshly dug fruit at a frequency of 79% and 52%, respectively. The percentage of infestation was reduced by drying in the field. This was particularly true of the inverted windrow. The proportion of shells and seed infested with a microorganism was reduced 13% and 36%, respectively, after field drying for 5 to 7 days in random and inverted windrows. Microorganisms were isolated much more frequently from shell pieces (73%) than from seed (36%).     

Additive and Synergistic Growth-inhibiting Properties of the Canaline-Urea Cycle Amino Acids

Growth studies with Lemna minor revealed the additive and synergistic growth-inhibiting properties of the canaline-urea cycle amino acids. Simultaneous canavanine and canaline treatment caused an additive reduction in frond production. Ureidohomoserine interacted with canaline or canavanine to affect synergistically L. minor growth by enhancing individual canavanine or canaline toxicity and increasing the additive growth reduction caused by canavanine plus canaline. The ornithineurea cycle amino acids effectively counteracted both the additive and synergistic growth-inhibiting properties of the canaline-urea cycle compounds.     

Pollen and resource limitations to lifetime seed production in a wild population of the endangered plant Disanthus cercidifolius var. longipes H. T. Chang (Hamamelidaceae)

Disanthus cercidifolius Maxim. var. longipes H. T. Chang, a plant species that only occurs in a few counties in Hunan, Jiangxi and Zhejiang Provinces and with a relatively small number of individuals, is ranked as a second Class endangered species for conservation in China. We have studied the effect of pollen and resources available to female reproduction, and the reproductive mechanism of “excess flowers with low fruit set” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang was discussed. Results are as follows Pollen from different sources has significant effects on fruit set and seed set of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. The pollen source rather than pollen numbers significantly affected reproduction of this species. In wild populations, producing one fruit needs about 54.8 flowers, and one satiation seed needs about 6.60 flowers or 83.19 ovules. After fertilizing, which was propitious to flower development, the abortion rate of flower buds was decreasing, but the flowering rate was increasing. The fruit set and seed set was also significantly increasing, while abortion rate of fruit was significantly decreasing. With the increasing percentages of cutting leaves, the fruit set decreased, but the abortion rate of fruit shows no significant differentiation among treatments. After cutting branches that were puny, broken and insectin-fested branches, the flower number seemed to be decreasing, but the fruit set and seed set all increased significantly. After removing some flowers, the fruit set was calculated with respect to the number of flowers remaining after the treatment increased with increasing of percentages of flower removal, whereas fruit set calculated with respect to the initial number of flowers remained constant, and the mean weights of per fruit and per seed all decreased significantly. Sufficient spatial or temporal heterogeneities in nutrient levels might allow limitation of seed set by resources and pollen in a natural population, while supplying resources may indirectly affect pollination by increasing attraction of the flowers to pollinators. There were very low fruit and seed sets in natural populations of Disanthus cercidifolius Maxim. var. longipes H. T. Chang. Different factors may have interacted to effect a low fruit set. A joint adoption of the “selection abortion hypothesis”, “ovary reserve hypothesis” and “male function hypothesis” seems to be the most likely explanation for the reproductive strategy of “excess flowers with few fruit sets” in Disanthus cercidifolius Maxim. var. longipes H. T. Chang.     

Further studies of the effect of L-canavanine on the tobacco hornworm, Manduca sexta.

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of ¹⁴C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.     

Anthropogenic edges,isolation and the flowering time and fruit set of Anadenanthera peregrina,a cerrado savanna tree

Fragmentation exposes plants to extreme environmental conditions with implications for species phenology and reproduction. We investigated whether isolation and edge effects influence size, flowering time, fruit set, and seedling establishment of Anadenanthera peregrina var. falcata. We compared trees in the interior (n?=?85), and on the edge (n?=?74) of a cerrado savanna fragment as well as in a pasture (n?=?26) with respect to size, flowering phenology, flower and fruit production, fruit and seed set, predispersal seed predation, and seedling establishment. Trees in the pasture were larger and produced a higher number of flowers and fruits than trees on the edge and interior, yet seed set did not differ across environments. The plant size structure explained the flower and fruit production, and the self-compatibility breeding system caused a similar seed set regardless of the environment. First flowering was later and fruit set higher in the interior. We argue that time of first flower influenced the fruit set of Anadenathera. Edge and isolated trees started to flower earlier as a response to microclimatic conditions—mainly temperature—reducing the fruit set. Predispersal seed predation was lower among pasture trees. Conversely, we found seedlings only on the edge and in the interior of cerrado, suggesting that the pasture was of poor quality habitat for Anadenanthera recruitment. Isolation affected the plant size structure and reproduction of Anadenanthera trees. Studies comparing plant phenology under contrasting environmental conditions may offer clues on how global change may affect plant reproduction in the tropics.     

The role of the persistent fruit wall in seed water regulation in Raphanus raphanistrum (Brassicaceae)

Background and Aims

Dry fruits remain around the seeds at dispersal in a number of species, especially the Brassicaceae. Explanations for this vary, but usually involve mechanisms of innate dormancy. We speculate that, instead, a persistent fruit may give additional protection through control of dehydration, to species growing in arid or Mediterranean environments where water is sporadic.

Methods

X-rays and weight measurements were used to determine the extent to which Raphanus raphanistrum seeds within mature fruits imbibe water, and germination tests determined the roles of the fruit and seed coat in seed dormancy. Rates of water uptake and desiccation, and seedling emergence were compared with and without the fruit. Finally, germinability of seeds extracted from fruits was determined after various periods of moist conditions followed by a range of dry conditions.

Key Results

Most seeds rapidly take up water within the fruit, but they do not fully imbibe when compared with naked seeds. The seed coat is more important than the dry fruit wall in maintaining seed dormancy. The presence of a dry fruit slows emergence from the soil by up to 6–8 weeks. The fruit slows the rate of desiccation of the seed to a limited extent. The presence of the fruit for a few days during imbibition somehow primes more seeds to germinate than if the fruit is absent; longer moist periods within the pod appear to induce dormancy.

Conclusions

The fruit certainly modifies the seed environment as external conditions change between wet and dry, but not to a great extent. The major role seems to be: (a) the physical restriction of imbibition and germination; and (b) the release and then re-imposition of dormancy within the seed. The ecological significance of the results requires more research under field conditions.     

Impact of habitat structure and fruit abundance on avian seed dispersal and fruit predation

So far, it is poorly understood how differential responses of avian seed dispersers and fruit predators to changes in habitat structure and fruit abundance along land-use gradients may translate into consequences for the seed dispersal of associated plants. We selected a gradient of habitat modification (forest, semi-natural, and rural habitat) characterized by decreasing tree cover and a high variation in local fruit availability. Along this gradient we quantified fruit removal by avian seed dispersers and fruit predators from 18 Sorbus aucuparia trees. We analyzed the relative importance of tree cover and fruit abundance in explaining species richness, abundance and fruit removal rates of both guilds from S. aucuparia trees. Species richness and abundance of seed dispersers decreased with decreasing tree cover, whereas fruit removal by seed dispersers decreased with decreasing fruit abundance independent of tree cover. Both variables had no effect on species richness, abundance and fruit removal by fruit predators. Consequently, seed dispersers dominated relative fruit removal in fruit-rich sites but the dispersal/predation ratio shifted in favor of predation in fruit-poor habitat patches. Our study demonstrates that variation in local habitat structure and fruit abundance can cause guild-specific responses. Such responses may result in a shift in fruit removal regimes and might affect the dispersal ability of dependent fruiting plants. Future studies should aim at possible consequences for plant recruitment and guild-specific responses of frugivores to disturbance gradients on the level of entire plant–frugivore associations.     

Seed size variation and its effect on germination and seedling performance in the clonal herb Convallaria majalis

Variation in seed size is common both within and among plant species. This study examined within-species variation in seed weight, and its implications for some components of fitness in the clonal herb Convallaria majalis. This species produces berries containing 4.1 seeds on average. The average seed weight was 16.5 mg, with a coefficient of variation of 32.7%. Seed packaging in fruits was on average 12.5%, and showed a slight tendency to increase with fruit weight. A trade-off was found between seed weight and seed number both within fruits and within ramets. The probability and timing of germination was not influenced by seed size. A field experiment and indirect evidence suggested that post-dispersal seed predation was not related to seed size. Increasing seed weight conferred an advantage to developing seedlings. This advantage was enhanced if a seedling was growing in the close vicinity of a seedling of another species. It is suggested that seed size variation in C. majalis primarily is the result of resource variation during fruit development. A conflict between parents and offspring may however contribute to increase seed size variation.     

Ecosystems effects 25 years after Chernobyl: pollinators, fruit set and recruitment

Animals are assumed to play a key role in ecosystem functioning through their effects on seed set, seed consumption, seed dispersal, and maintenance of plant communities. However, there are no studies investigating the consequences of animal scarcity on seed set, seed consumption and seed dispersal at large geographical scales. We exploited the unprecedented scarcity of pollinating bumblebees and butterflies in the vicinity of Chernobyl, Ukraine, linked to the effects of radiation on pollinator abundance, to test for effects of pollinator abundance on the ecosystem. There were considerably fewer pollinating insects in areas with high levels of radiation. Fruit trees and bushes (apple Malus domestica, pear Pyrus communis, rowan Sorbus aucuparia, wild rose Rosa rugosa, twistingwood Viburnum lantana, and European cranberry bush Viburnum opulus) that are all pollinated by insects produced fewer fruit in highly radioactively contaminated areas, partly linked to the local reduction in abundance of pollinators. This was the case even when controlling for the fact that fruit trees were generally smaller in more contaminated areas. Fruit-eating birds like thrushes and warblers that are known seed dispersers were less numerous in areas with lower fruit abundance, even after controlling for the effects of radiation, providing a direct link between radiation, pollinator abundance, fruit abundance and abundance of frugivores. Given that the Chernobyl disaster happened 25 years ago, one would predict reduced local recruitment of fruit trees if fruit set has been persistently depressed during that period; indeed, local recruitment was negatively related to the level of radiation and positively to the local level of fruit set. The patterns at the level of trees were replicated at the level of villages across the study site. This study provides the first large-scale study of the effects of a suppressed pollinator community on ecosystem functioning.