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.     

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.     

An Ontogenetic Study of Canavanine Formation in the Fruit of Jack Bean, Canavalia ensiformis (L.) DC

An ontogenetic study of canavanine formation in the fruit of jack bean, Canavalia ensiformis (L.) DC. was conducted. Evidence was presented to show that the ovary wall is the reservoir for seed canavanine. The testa possesses sufficient canavanine to account for the continued elevation in seed canavanine after the pod senesces. The seed canavanine concentration is not constant inasmuch as the canavanine content per milligram dry weight or soluble protein increases abruptly with seed growth and levels off only with the onset of fruit ripening.     

Investigations of Canavanine Biochemistry in the Jack Bean Plant, Canavalia ensiformis (L.) DC: II. Canavanine Biosynthesis in the Developing Plant

The canavanine content of developing leaves of jack bean, Canavalia ensiformis (L.) DC., increases during leaf development. The leaf possesses the enzymes required for synthesizing canavanine by a cyclic series of reactions analogous to the ornithine-urea cycle. This reaction series involves the sequential formation of canaline, O-ureidohomoserine, and canavaninosuccinic acid.     

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.     

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.     

Canavanine metabolism in tissue cultures of Canavalia lineata

The greening of callus was achieved by modulating the medium's growth regulator concentrations under continuous light. Canavalia lineata (L.) DC. calluses formed chlorophyll when they were exposed to continuous light in the presence of benzylaminopurine and indole-3-acetic acid. Canavanine and canaline were detected in the green callus. But only canaline was detected in the white callus grown in the dark. Feedings of canaline to suspension cultures showed that the green suspended cells were capable of de novo biosynthesis of canavanine, but the white suspended cells were not. Exogeneously supplied canavanine was used to produce canaline and homoserine by the white suspended cells. Arginase activity was induced by the addition of arginine or canavanine to the medium, and canaline reductase activity was induced by the addition of canaline but not with ornithine in the white suspended cells.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - OPA o-phthaldialdehyde - PC Phillips & Collins (1979) medium     

Effects of Canavanine on Arginine Utilization in Plants with Special Reference to Mitotic Activity

Excised primary roots of Phaseolus vulgaris L. were treated with canavanine and the effect on arginine utilization was studied. Arginine utilization was observed to be depressed to a low level by canavanine (1.8 × 10^?4M) in rapidly dividing and growing tissues. In addition, canavanine inhibited arginine utilization to a lesser degree in root sections composed of mature and non-dividing tissues. This demonstrated that canavanine inhibition is not limited to those tissues where active DNA synthesis occurs. Canavanine was observed to inhibit the onset of mitosis in primary roots as evidenced by a significant reduction of the frequency of mitotic figures. In addition, this amino acid was found to affect the course of mitosis once it was initiated by altering the relative frequency of mitotic stages, decreasing the percentage of prophase figures and increasing the percentage observed in the telophase. An influence on the rate of spiralization and despiralization of the chromosomes is suggested. The effects on mitosis are probably related to growth-inhibitory and toxic effects of canavanine. The possible function of canavine as an allelopathic substance is hypothesized.     

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.     

A higher plant enzyme exhibiting broad acceptance of stereoisomers

An arginase, purified from the leaf of the jack bean, Canavalia ensiformis, can effectively hydrolyze both l- and d-arginine. Arginases, examined from a number of other plant and animal sources, exhibit marked substrate stereospecificity and fail to catabolize d-arginine. In order to provide essential nitrogen, jack bean leaf arginase also catabolizes l-canavanine, an arginine analog that is a predominant nitrogen-storing metabolite of this legume. The ability of arginase to metabolize both stereoisomers of arginine may result from the requirement for this enzyme to exhibit limited substrate specificity in order to hydrolyze both arginine and canavanine.     

Variation in the Amino Acid Concentration During Development of Canavalia ensiformes

The distribution of amino acids in distinct tissues of Canavalia ensiformes was determined during the life cycle of the plant. Glycine was shown to be the main amino acid in mature seeds, while the nonprotein amino acid canavanine exhibited a high concentration in 7-d-old seedlings. Canavanine was lower in the seeds when compared to other tissues analyzed. This does not support the nitrogen-storage function of canavanine, however, it suggests that it is involved in the translocation of amines during the early stages of the development. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the Inhibitory Effect of Canavanine on the Replication of Influenza RI/5+ Virus: II. Interaction of M Protein with the Plasma Membrane

When influenza A/RI/5⁺ virus-infected cells were incubated in medium to which 2 μg of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding-like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine-treated cells by freeze-thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine-substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5⁺, because of the inability of M protein to associate with the plasma membrane.     

Canavanine synthesis in thein vitro propagated tissues ofCanavalia lineata

Maximum shoot induction from stem explants ofCanavalia lineata was obtained with an agar-solidified PC medium containing 10 M benzylaminopurine and 1 M naphthaleneacetic acid. Rooting of thesein vitro produced shoots was achieved with hormone-free PC medium. Canavanine was produced almost exclusively in the leaves and was not detected in the roots ofin vitro propagatedC. lineata. To exclude the possibility of imminent translocation of canavanine from the root to leaf, adventitious roots were induced from leaf explants in PC medium supplemented with 1 M kinetin and 20 M indole-3-acetic acid and subcultured in medium lacking growth regulators, and the roots excised from germinated seedlings were cultured in hormone-free PC medium. All the roots were incapable of accumulation of canavanine. These results suggest that leaves ofC. lineata are the possible site of canavanine synthesis.     

l-Canavanine Transport and Utilization in Developing Jack Bean, Canavalia ensiformis (L.) DC. [Leguminosae

l-Canavanine, the guanidinooxy structural analog of l-arginine, is an important nonprotein amino acid of many leguminous plants with nitrogen storage a major proported role. l-[Guanidinooxy-¹⁴C]canavanine, [¹⁴C] urea, and [¹⁵N]urea were injected separately into the fleshy, green cotyledons of 9-day old jack bean plants, Canavalia ensiformis (L.) DC. [Leguminosae]. There was significant transport of canavanine from the cotyledons to the aboveground portions of the plant, but not to the roots. Within 1.5 hours of isotope administration, the remaining labeled canavanine was divided equally between the cotyledons and the aboveground portions of the plant. During the 48-hour postinjection period, the contribution of l-[guanidinooxy-¹⁴C]canavanine to the total ¹⁴carbon of the cotyledons decreased rapidly while it increased in the aboveground portions of the plant.     

The biological effects and mode of action of L-canavanine, a structural analogue of L-arginine.

Many of the 200 or so non-protein amino acids synthesized by higher plants are related structurally to the constituents of common proteins. L-Canavanine, the guanidinooxy structural analogue of L-arginine, is representative of this group. It has provided valuable insight into the biological effects and the mode of action of non-protein amino acids which acts as analogues of the protein amino acids. The arginyl-tRNA synthetases of numerous canavanine-free species charge canavanine, and canavanine is subsequently incorporated into the nascent polypeptide chain. Production of canavanine-containing proteins ultimately can disrupt critical reactions of RNA and DNA metabolism as well as protein synthesis. Canavanine also affects regulatory and catalytic reactions of arginine metabolism, arginine uptake, formation of structural components, and other cellular precesses. In these ways, canavanine alters essential biochemical reactions and becomes a potent antimetabolite of arginine in a wide spectrum of species. These deleterious properties of canavanine render it a highly toxic secondary plant constituent that probably functions as an allelochemic agent that deters the feeding activity of phytophagous insects and other herbivores.     

Canavanine incorporation into the antibacterial proteins of the fly, Phormia terranovae (Diptera), and its effect on biological activity

In response to microbial infection or mechanical injury, larvae of the fly, Phormia terranovae (Diptera), can induce de novo production of a group of antibacterial proteins including: peak I protein, diptericin A, diptericin B, diptericin C, and peak V protein. Administration of L-canavanine at the time of mechanical injury results in the incorporation of this arginine antagonist into these proteins. Canavanine replacement for arginine causes a total loss of detectable antibacterial activity for diptericin B and diptericin C, whereas diptericin A and peak V protein are severely inhibited. This loss in biological activity occurs in spite of the fact that canavanine stimulates induced protein synthesis. Analysis of the hydrolysate of diptericin A reveals that one-third of the 3 arginyl residues are replaced by canavanine. This investigation provides the first evidence that canavanine incorporation into a protein can impair its function.     

Narbonin,a novel 2S protein from Vicia narbonensis L. seeds: cDNA,gene structure and developmentally regulated formation

cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M _r ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.     

Canavanine-lnduced inhibition of growth and heterocyst differentiation inAnabaena doliolum and isolation of a canavanine-resistant mutant

The effect of the arginine analogue, canavanine on growth and heterocyst differentiation in the nitrogen-fixing algaAnabaena doliolum has been studied. The analogue inhibited growth and heterocyst differentiation at a concentration as low as 1 μM. The treated algal cells lacked conspicuous granular inclusions, whereas treatment with chloramphenicol led to increased synthesis of granules (probably cyanophycin granules). Exogenously added arginine completely reversed the effect of the analogue but lysine could only partially relieve the effect. A time course study with canavanine indicated inhibition of fresh protein(s) synthesis at all steps where a new class of proteins is synthesized so that the action of the analogue does not seem to be specific for a particular kind of protein. A mutant resistant to this analogue has been successfully isolated indicating that this alga does not show mutational immunity at least to the amino acid analogues unlike in the observation with different antibiotics. Our observations indicate that canavanine either directly inhibits protein synthesis or forms defective protein(s) which produces all the observed effects.     

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.