Vicia sativa L. 'Run-off' and Purified Ribosomes: Polyphenylalanine Synthesis and Molecular Action of Ribosome-inactivating Proteins

A ‘run-off’ cell-free translation system (a 30000x g supernatant; S 30) has been prepared from 4–5-d-oldembryonic axes of Vicia sativa L., a plant lacking ribosome-inactivatingprotein activities which is very sensitive to certain RIPs butnot to others. The system was able to generate a high rate ofpolyphenylalanine synthesis upon addition of polyuridylic acid.From this supernatant, purified ribosomes and a 100000 x g supernatantwere prepared which were able to perform polyphenylalanine synthesiswhen mixed together (reconstituted system). The most importanttranslation parameters were optimized in each case. Both theS 30 and the reconstituted system displayed differential sensitivitiesto certain RIPs. The purified RIP-inactivated ribosomes wereable to release a 370 nucleotide rRNA fragment diagnostic forRIPs upon treatment of the isolated rRNA with acid aniline. Key words: Translation, polyphenylalanine synthesis, ribosomes, ribosome-inactivating proteins, Vicia sativa     

Some Effects of Temperature and Substrate Content upon Respiration and the Carbon Balance of Field Beans (Vicia faba L.)

The effects of substrate content and temperature upon the productionof carbon dioxide in the dark were investigated in Vicia fabaand Sorghum vulgare, using the time courses for carbon dioxiderelease. No endogenous rhythms were found. With V. faba at highsubstrate contents, a steady rate of respiration was measuredat low temperatures, suggesting that the rate of respirationis limited by a third factor such as enzyme activity. This steadyrate eventually decreased rapidly. There was a first order rateof decrease with time at higher temperatures. Low substratecontents gave a complex decay. The rate of decrease of the rateof respiration was affected by the initial state of the plantsover the same range of rates. Reasons for this are discussed.The temperature sensitivity of respiration was investigated.The respiration of plants with high substrate contents had alower temperature sensitivity than those with low substratecontents. This was further investigated by measuring the stoichiometryof carbon dioxide production in the dark from total solublecarbohydrate (as hexose equivalent). It is likely that incompletehexose respiration, which occurs at high substrate contents,is less temperature sensitive than complete hexose respirationor the respiration of some other substrate. Vicia faba, Sorghum oulgare, carbon dioxide, respiration, temperature, substrate content     

A Cucumis sativus cell-free translation system: preparation, optimization and sensitivity to some antibiotics and ribosome inactivating proteins

A cell-free translation system was prepared from 3- to 5-day-old embryonic axes of gherkin ( Cucumis sativus L.). The system was optimized for Mg²⁺, K⁺, NH⁺₄, high speed supernatants, tRNA mixture from wheat germ, time and temperature. The system translates efficiently both endogenous mRNA (using a 30000 g supernatant) and polyuridylic acid (using either a 30000 g supernatant or a 100000 g supernatant supplemented with purified ribosomes). Translation by gherkin ribosomes was inhibited by several well-known eukaryotic inhibitors, antibiotics and ribosome-inactivating proteins. A translational inhibitory activity found in Cucumis sativus L. dry seeds acted on polypeptide synthesis carried out by cell-free systems from several mammals and plants, including gherkin embryonic axes. Our results indicate that the inhibitor is located in the seed bark and cotyledons, and is either blocked or absent in the embryonic axes, thus allowing the isolation of active gherkin ribosomes. The presence of the putative inhibitor appeared to be unevenly distributed in developing plants.     

Elderberry (Sambucus nigra L.) seed proteins inhibit protein synthesis and display strong immunoreactivity with rabbit polyclonal antibodies raised against the type 2 ribosome-inactivating protein nigrin b

Affinity chromatography-purifled elderberry (Sambucus nigraL.) seed proteins strongly inhibited protein synthesis and displayedthe 28S rRNA N-glycosidase activity characteristic of all typesof ribosome-inactivating proteins (RIPs). Western blot analysisrevealed several proteins that reacted with antibodies raisedagainst the novel non-toxic type 2 ribosome-inactivating proteinnigrin b isolated from elder bark, thus indicating the presenceof a new type 2 RIP. Key words: Anti-nigrin b antibodies, protein synthesis, seeds, elder seeds, Sambucus nigra     

In situ observations of stomatal movements in different light-dark regimes: the influence of endogenous rhythmicity and long-term adjustments

The technical device for continuous microscopic observationsof stomatal movements in a gas exchange chamber using digitalimage analysis, earlier described by Kappenet al. (1994), wastechnically improved. By electronic remote control, it is nowpossible repeatedly to record over a period of several daysidentical stomata on a 25 50 mm leaf area. The responses ofindividual stomata to various light-dark sequences in the lightand in the dark phase were investigated on attached leaves ofViciafaba (L.). The amplitude of stomatal oscillations caused byalternatinglight was highest on the first day of an experimental seriesand decreased with repeated application, indicating a long-termadjustment to changing light conditions by decreasing sensitivityto intermittent darkening. In the usual dark phase light-darksequences had little effect on the stomatal aperture, whichremained small. These results were widely convergent with thestomatal conductance calculated from the gas exchange measurements.Very prominent is the role of an endogenous rhythmicity of thestomatal response. It could be demonstrated (1) by autonomousstomatal opening before the light phase started; (2) by a suppressionof dark response in the early morning hours; (3) by a decreasinglight stimulation in the afternoon; (4) by further increaseof aperture for several hours if no light was provided in thelight phase. Closely adjacent stomata could show divergent dark-openingmovements pointing to an autonomous control mechanism locatedin the guard cells. The endogenously controlled morning openingprovides full assimilation capacity in the usually humid morninghours when transpiratory water loss associated with C02-uptakeis comparatively small. Key words: Stomata, changing PPFD, desensitization, endogenous rhythmicity, diurnal courses, Vicia faba     

Effect of Osmotic Stress Upon Endogenous Hormone Levels in Euphorbia lathyrus L. and Vicis faba L.

Ethylene concentrations rise in stems, lacunae and roots ofEuphorbia Iathyrus subjected to osmotic stress. In contrastto other species, however, no such changes are observed in leaves.Similarly, ABA concentrations rise in the leaves of osmotically-stressedplants but little change was observed in roots. In no case wasthere any strong evidence for the existence of a threshold ofosmotic potential above which a response will not occur. Instead,marked increases in concentrations of both ABA and ethylenewere obtained in response to osmotic stresses which did notresult in significant changes in water saturation defecit (WSD)during the experiment. In Vicia faba, time course studies at constant osmotic potentialshowed that ABA concentrations increased with duration of stressand increased WSD but the effect was most marked in youngertissues for a given level of stress. Concentrations of boundABA showed a similar pattern with time but here the change wasleast marked in the younger tissues. Euphorbia Iathyrus, Vicia faba, endogenous growth regulators, osmotic stress     

Sucrose and Hexose Release by Excised Stem Segments of Vicia faba L.: The Sucrose-Specific Stimulating Influence of Cuscuta on Sugar Release and the Activity of Acid Invertase

By washing out solutes in 0.5 mM CaSO⁴at 25 °C during aperiod of 5–6 h, the release of sugars by excised stemsegments of Vicia faba L. was measured. The stem parasite Cuscutaeuropaea strongly stimulated the release of sucrose into theefflux medium; this effect was most marked during the last hoursof each experiment but this stimulating effect of the parasitecould not be detected for glucose and fructose. The fact thatparasitized stem segments released higher than normal hexoseamounts during the last hours of several experiments, couldbe explained as the result of extracellular hydrolysis of sucroseby free space invertase. A high free space acid invertase activitywas present in young stem segments of Vicia faba and in tissuesof Cuscuta. The stimulating influence of Cuscuta on sugar releaseby cells of stem segments appears sucrose-specific, supportingthe idea that the stimulating influence of Cuscuta on sugarrelease is restricted to the sieve-tube system. When metabolic inhibitors were added to the washing solutionor when segments were incubated at low temperature, no cleareffect of the parasite could be observed and for all segments(parasitized and non-parasitized) a strongly enhanced releaseof sucrose into the efflux medium was found during the lasthours of an experiment. These data support the idea that anintensive resorption of sucrose occurs within stem segments,after its release into the apoplast. Key words: Cuscuta europaea, Parasitic relationship, Phloem unloading     

Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations.     

The effect of salinity on N fixation and assimilation in Vicia faba

Production of the faba bean in semi-arid and coastal areas maybe limited by the salt sensitivity of faba bean symbiosis. Accordingly,this study was done to analyse the effects of salt on the effectivesymbiosis of faba bean (Vicia faba L. var. minor cultivar Alborea)and salt-tolerant Rhizobium leguminosarum biovar. viciae strainGRA19. After 4 weeks of growth, the nutrient solutions weresupplemented with 50, 75 and 100 mM NaCl for 21 d. Plants wereharvested four times at weekly intervals, beginning at 4 weeks.Vicia faba tolerated low (50 mM NaCl) but not higher levels(75 and 100 mM NaCl) of salt stress. Salinity affected shootgrowth more than root growth. At the end of the culture, thetotal nitrogen content in the shoot was affected more than plantgrowth; conversely, in the root, growth was influenced morethan total nitrogen content. In nodules, nitrogen fixation (acetylenereduction activity) was more sensitive to salinity than ammoniumassimilation (glutamine synthetase and glutamate synthase). Key words: Glutamate synthase, glutamine synthetase, N₂ fixation, Rhizobium leguminosarum, salinity     

Total Uptake and Incorporation into DNA of 3H-Thymidine, 3H-Deoxyuridine and 3H-Thymine in the Primary Root of Vicia faba L.: I. INTACT ROOTS

Total uptake and incorporation of ³H-thymidine, ³H-thymine,and ³H-deoxyuridine into DNA have been investigated in the apical3 cm of the primary root of Vicia faba. Evidence has been obtainedthat endogenous TdR in these roots may be transported eitherapically or basally; apical movement being greater than movementfrom the apex towards the base of the root. The results havebeen discussed with respect to the possible distribution ofendogenous pools of thymidine, thymine, and deoxyuridine inthe primary root of V. faba.     

Cellular pathway of photosynthate transport in coats of developing seed of Vicia faba L. and Phaseolus vulgaris L. I. Extent of transport through the coat symplast

The extent of post-phloem solute transport through the coatsymplasts of developing seeds of Vicia faba L. and Phaseolusvulgaris L. was evaluated. For Vicia seed coats, the membrane-impermeantfluorochrome, CF, moved radially from the chalazal vein to reachthe chlorenchyma and thin-walled parenchyma transfer cell layers.Thereafter, the fluorochrome moved laterally in these two celllayers around the entire circumference of the seed coat. Transferof CF from the chalazal vein was inhibited by plasmolysis ofattached ‘empty’ seed coats. In contrast, the spreadof phloem imported CF was restricted to the ground parenchymaof Phaseolus seed coats. Fluorochrome loaded into the outermostground parenchyma cell layer was rendered immobile followingplasmolysis of excised seed-coat halves. Phloem-imported [¹⁴C]sucroseand the slowly membrane permeable sugar, L-[¹⁴C]glucose, werepartitioned identically between the vascular and non-vascularregions of intact Vicia seed coats. For ¹⁴C-photosynthates,these partitioning patterns in attached ‘empty’Vicia seed coats were unaffected by PCMBS, but inhibited byplasmolysis. Tissue autoradiographs of intact Phaseolus seedcoats demonstrated that a pulse of ¹⁴C-photosynthate moved fromthe veins to the grounds tissues. In excised Vicia seed coats,preloaded with ¹⁴C-photosynthates, the cellular distributionof residual ¹⁴C-label was unaffected by PCMBS. In contrast,PCMBS caused the ¹⁴C-photosynthate levels to be elevated inthe veins and ground parenchyma relative to the branch parenchymaof Phaseolusseed coat halves. Based on the above findings, itis concluded that the phloem of Vicia seed coats is interconnectedto two major symplastic domains; one comprises the chlorenchyma,the other the thin-walled parenchyma plus thin-walled parenchymatransfer cells. For Phaseolusseed coats, the phloem forms amajor symplastic domain with the ground parenchyma. Key words: Phaseolus vulgaris L, phloem unloading, photosynthate transport, seed coat, symplast, Vicia faba L     

Resistance to Water Loss from the Mesophyll Cell Surface in Plant Leaves

A method is described for the accurate measurement of the apparentresistance to water loss from the mesophyll cell walls of plantleaves (r_w), and for studying the mechanism underlying thisresistance. The method for distinguishing possible mechanismsinvolves a comparison of the calculated values of r_w at differentrates of evaporation. The value of r_w remained below 50 s m^–1at relative water contents greater than 11 ± 3% and 7± 2% for Pelargonium hortorum Bailey and Vicia faba L.respectively. Therefore r_w is relatively insignificant at normalphysiological water contents in these species. When r_w did increaseit was not sensitive to evaporation rate, suggesting that alowering of the vapour pressure at the evaporating sites wasnot involved. This contrasted with the results for cellulosefilter paper, where r_w was more sensitive to evaporative flux.     

Increased accumulation of trichosanthin in Trichosanthes kirilowii induced by microorganisms

Trichosanthin, a type 1 ribosome-inactivating protein, is highlyexpressed in the root tuber of Trichosanthes kirilowii whengrown under normal greenhouse conditions. The expression levelof trichosanthin was significantly reduced when the seeds weregerminated and subsequently grown in a sterile environment.However, co-cultivation of the sterile T. kirilowii with microorganismsresults in an accumulated level of trichosanthin suggestinga possible role of trichosanthin in defence against pathogens. Key words: Trichosanthin, Trichosanthes kirilowii, expression levels, fungi     

Inhibitory effect of lycorine on cell division and cell elongation

Lycorine, an alkaloid isolated from bulbs of Amarillidaceae,was found to be a powerful inhibitor of cell division and elongation.Adding different concentrations of lycorine from 10^–6M to 10^–4 M in an appropriate growth-medium strongly inhibitedcell division in explants of lettuce pith parenchyma. The sameresult was obtained with liquid yeast cultures growing exponentially. Lycorine-treated meristematic cells of the primary roots ofVicia faba also showed rapid inhibition of the mitotic indexwhile interphase cells increased proportionately. Lycorine alsoinhibited endogenous and auxin-induced cell elongation in Avenacoleoptiles and pea segments. Since both cell division and cell elongation require proteinsynthesis and RNA synthesis, the assumption is that lycorineprobably inhibits one of the two syntheses. ¹This study was supported by a contract between the NationalResearch Council of Italy and University of Bari, Instituteof Botany. (Received November 27, 1972; )     

Trichosanthin, alpha-momorcharin and beta-momorcharin: identity of abortifacient and ribosome-inactivating proteins

The abortifacient proteins trichosanthin, alpha-momorcharin and beta-momorcharin at nM concentrations inhibit cell-free protein synthesis. The momorcharins and the ribosome-inactivating proteins isolated from Momordica charantia seeds cross-react with the respective antisera. The ribosome-inactivating proteins saporins, pokeweed antiviral protein (PAP) and, to a lesser extent, gelonin have abortifacient activity on pregnant mice.     

Protein synthesis in the embryo of Pinus thunbergii seed II. An induction by light

¹⁴C-Amino acid incorporating activity in the absence of exogenousmRNA was found in a cell-free system from embryos of light-germinatedPinus thunbergii seeds, but not in that from dark-imbibed seedembryos. Template activity in the cell-free system from thelight-germinated seed embryos was observed in the ribosome fraction,especially the polyribosome fraction, but not in the 100,000?gsupernatant fraction (s100). These facts suggest that the natureof the block in protein synthesis during the imbibition of seedsin the dark is due to the lack or inactivity of mRNA. The s100from light-germinated seed embryos was found to be less activein amino acid incorporation than that from dark-imbibed seedembryos. (Received November 29, 1976; )     

Transformation of Vicia faba Cotyledon and Stem Tissues7Agrobacterium rhizogenes: Infectivity and Cytological Studies

Transformation of Vicia faba cotyledon and stem tissues by Agrobacteriumrhizogenes has been demonstrated. Hormone autotrophy and NPTIIdot blot assays were used to confirm the transgenic nature ofroot clones, and to show the co-transfer of the T-DNA from theRi plasmid and the NPTII gene from pBIN 19. An effect of plantgenotype on susceptibility to A. rhizogenes was observed. Theresponse to infection depended on tissue type in addition toplant genotype. Cytological analysis of transformed root clonesuncovered variation in ploidy up to the octoploid level andstructural rearrangements of chromosomes. The possible originsof these karyotype changes and their implications are discussed. Key words: Vicia faba, Agrobacterium rhizogenes, transformed roots, genotypic effects, chromosomes     

In vitro production of dianthin from crown gall lines of carnation (Dianthus caryophyllus L.)

Tumorous crown gall lines of carnation (Dianthus caryophyllus L.) were obtained by transforming leaf explants by the co-cultivation procedure; transformed lines were checked for their ability to produce in vitro the type 1 ribosome-inactivating proteins dianthins. Crude extracts from cultured callus were able to inhibit protein synthesis in a cell-free system based on rabbit reticulocyte lysate. A protein with an apparent molecular mass comparable to that of dianthin 30 from leaves was identified from its chromatographic behaviour and by Western analysis. Dianthin from callus showed a specific activity comparable to that reported for the leaf isoform. The presence and accumulation in the tumorous line of a 37 kDa protein with no ribosome-inactivating activity and strong antigenic reactivity to dianthins is discussed. Received: 18 July 1997 / Revision received: 10 January 1998 / Accepted: 5 February 1998