Effects of Glyphosate on Caffeic Acid Metabolism in Perilla Cell Suspension Cultures

Perilla cell suspension cultures contained caffeic acid at 0.073%of the fresh weight. When 1 min glyphosate was administeredto the cell culture in the logarithmic and stationary phases,the amount of caffeic acid ceased to increase and remained ata nearly constant level during the following several days. Phenylalanineammonia-lyase (PAL) activity in the cell culture increased immediatelyafter transfer to a fresh medium, decreased rapidly in the midstof the logarithmic phase and became almost undetectable in thestationary phase. PAL activity was markedly inhibited by glyphosateat more than 0.2 mM. The shikimic acid content of cells from14-day culture grown in the presence of 1 mM glyphosate increasedup to 74.9 µg per g fresh weight during 6-day culture,whereas that of the control cells was undetectable. The dosageof 0.15 mM L-2-aminooxy-3-phenylpropionic acid (L-AOPP), aninhibitor of PAL, to the cells did not cause shikimic acid accumulation. (Received June 25, 1983; Accepted October 6, 1983)     

Kinetics of Phenylalanine Ammonia-Lyase and the Effect of L-{alpha}-aminooxy-{beta}-phenylpropionic Acid on Enzyme Activity and Radicle Growth in Germinating Lettuce Seeds

The effects of different concentrations of L--aminooxy-ß-phenyIpropionicacid (AOPP), an analog of L-phenylalanine, on the activity ofphenylalanine ammonia-lyase (PAL, EC 4.3.1.5 [EC] ) and the growthof radicles in 24 h old germinating lettuce (Lactuca salivaL.) seeds were investigated. AOPP causes a significant inhibitionof PAL activity in the seeds (85% inhibition at 10⁴ M). It alsocauses a stimulation of radicle growth at that concentration.The results show that the inhibition of PAL by AOPP may be dueto an irreversible binding of the inhibitor to the enzyme leadingto its inactivation. AOPP also inhibits ethylene biosynthesisin germinating lettuce seeds which could probably explain thestimulation of radicle growth in these seeds. The enzyme shows typical Michaelis-Menten kinetics. The K_m forL-phenylalanine is 4.2 x 10⁵ M. The enzyme does not show anytyrosine ammonia-lyase activity. Various substrate analogs suchas D-phenylalanine, p-fluorophenylalanine, ß-phenyllacticacid, tryptophan and the product of the enzyme reaction, trans-cinnamicacid, inhibit the enzyme competitively. A number of intermediatesand endproducts of the phenylpropanpid pathway, except chlorogenicacid, do not show any inhibition. ¹Scientific contribution number 1423 from the New HampshireAgricultural Experiment Station. (Received May 9, 1986; Accepted September 8, 1986)     

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-α-aminooxy-β-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L--aminooxy--phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283–287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.Abbreviations CAH trans-cinnamate 4-hydroxylase - L-AOPP L--aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase     

Enzymological basis for herbicidal action of glyphosate

The effects of 1 millimolar glyphosate (N-[phosphonomethyl]glycine) upon the activities of enzymes of aromatic amino acid biosynthesis, partially purified by ion-exchange chromatography from mung bean seedings (Vigna radiata [L.] Wilczek), were examined. Multiple isozyme species of shikimate dehydrogenase, chorismate mutase, and aromatic aminotransferase were separated, and these were all insensitive to inhibition by glyphosate. The activities of prephenate dehydrogenase and arogenate dehydrogenase were also not sensitive to inhibition. Two molecular species of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase were resolved, one stimulated several-fold by Mn²⁺ (DAHP synthase-Mn), and the other absolutely dependent upon the presence of Co²⁺ for activity (DAHP synthase-Co). Whereas DAHP synthase-Mn was invulnerable to glyphosate, greater than 95% inhibition of DAHP synthase-Co was found in the presence of glyphosate. Since Co²⁺ is a V_max activator with respect to both substrates, glyphosate cannot act simply by Co²⁺ chelation because inhibition is competitive with respect to erythrose-4-phosphate. The accumulation of shikimate found in glyphosate-treated seedlings is consistent with in vivo inhibition of both 5-enolpyruvylshikimic acid 3-phosphate synthase and one of the two DAHP synthase isozymes. Aromatic amino acids, singly or in combination, only showed a trend towards reversal of growth inhibition in 7-day seedlings of mung bean. The possibilities are raised that glyphosate may act at multiple enzyme targets in a given organism or that different plants may vary in the identity of the prime enzyme target.     

O-Benzylhydroxylamine: An Inhibitor of Phenylpropanoid Metabolism in Plants

O-Benzylhydroxylamine (OBHA) is a potent inhibitor of phenylalanineammonialyase (PAL, EC 4.3.1.5 [EC] ) and phenylpropanoid metabolismas evidenced by its effects on three plant species [soybean(Glycine max (L.) Merr.), buckwheat (Fagopyrum esculentum Moench.),and mung bean (Vigna radiata L.)]. When supplied to roots, OBHA(10^–5 M) did not significantly inhibit light- or dark-growthof soybean seedlings, but reduced (25%) soluble hydroxyphenoliccompound accumulation in light-grown axes. Higher concentrations(510^–5 M) of OBHA caused reductions (25%) in axis freshweight of light-grown seedlings (72 h), but did not lower axisweight of dark-grown seedlings. Anthocyanin accumulation inhypocotyls of intact mung bean seedlings was reduced by 25%after 3 days light growth after treatment with OBHA (10^–5M) via root feeding. Anthocyanin content of excised, etiolatedbuckwheat hypocotyls floated on solutions of OBHA (10^–5M) and incubated in the light for 24 h was reduced by 40%. L-Phenylalanineand t-cinnamic acid, intermediates of phenylpropanoid metabolism,were able to partially reverse this inhibition in buckwheat.Extractable PAL activity (specific activity basis) in soybeanaxes was substantially reduced (20% in dark, 40% in light) asearly as 24 h after root feeding with OBHA (10^–5 M). Reductionof PAL activity (specific activity or per axis basis) by OBHAcompared to control levels, continued throughout a time courseof 96 h. Kinetic studies on soybean PAL revealed a K_m of 1.1mM for L-phenylalanine and an apparent K_i of 3.5 µM forOBHA. (Received May 31, 1985; Accepted August 6, 1985)     

The Effect of L-Phenylalanine on Phenylalanine Ammonia-lyase and Cell Division in Artichoke Callus Culture

5 x 10^–5 M L-phenylalanine overcame the inhibitory effectof white light on cell division in artichoke callus culturesand increased extractable phenylalanine ammonia-lyase (PAL)activity compared to cultures grown in the presence of 5 x 10^–4M phenylalanine The lower concentration of the amino acid alsoenhanced rates of uptake and incorporation of ¹⁴C labelled phenylalaninethroughout G₁ and S. Differences between the two concentrationswere greatest during S with a 4-fold increase in uptake anda 3-fold increase in incorporation It is suggested thereforethat the capacity of 5 x10^–5 M phenylalanine to offsetthe light effect is due to an indirect stimulatory effect onamino acid and protein metabolism Increased levels of extractablePAL activity would then be reflected by this general stimulationof protein synthesis. Helianthus tuberosus L, Jerusalem artichoke, callus culture, cell division, phenylalanine ammonia-lyase     

CHEMICAL CONTROL OF MORPHOGENESIS IN MOSS PROTONEMA

1. The protonema of the moss, Funaria hygrometrica, grows continuouslyin calcium-free liquid media.
2. The growth was promoted by additionof oxalate, although themorphogenesis resulting in formationof gametophytic buds onthe protonema was suppressed by theaddition.
3. Calcium oxalate promoted the growth of protonema,while at ahigh concentration (10^–2 M) it caused the formationofclumped protonema (falsebuds).
4. Addition of plant growthhormones, such as IAA, NAA, 2,4-D andgibberellin retarded thegrowth of protonema, while 2,4-D ata low concentration stimulatedthe growth of protonema.
5. Kinetin greatly stimulated the formationof gametophytic budsin the protonema, but these buds were foundto be morphologicallyand physiologically abnormal.

(Received January 29, 1965; )     

Glyphosate Inhibition of 5-Enolpyruvylshikimate 3-Phosphate Synthase from Suspension-Cultured Cells of Nicotiana silvestris

Treatment of isogenic suspension-cultured cells of Nicotiana silvestris Speg. et Comes with glyphosate (N-[phosphonomethyl]glycine) led to elevated levels of intracellular shikimate (364-fold increase by 1.0 millimolar glyphosate). In the presence of glyphosate, it is likely that most molecules of shikimate originate from the action of 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase-Mn since this isozyme, in contrast to the DAHP synthase-Co isozyme, is insensitive to inhibition by glyphosate. 5-Enolpyruvylshikimate 3-phosphate (EPSP) synthase (EC 2.5.1.19) from N. silvestris was sensitive to micromolar concentrations of glyphosate and possessed a single inhibitor binding site. Rigorous kinetic studies of EPSP synthase required resolution from the multiple phosphatase activities present in crude extracts, a result achieved by ion-exchange column chromatography. Although EPSP synthase exhibited a broad pH profile (50% of maximal activity between pH 6.2 and 8.5), sensitivity to glyphosate increased dramatically with increasing pH within this range. In accordance with these data and the pK_a values of glyphosate, it is likely that the ionic form of glyphosate inhibiting EPSP synthase is COO⁻CH₂NH₂⁺CH₂PO₃²⁻, and that a completely ionized phosphono group is essential for inhibition. At pH 7.0, inhibition was competitive with respect to phosphoenolpyruvate (K_i = 1.25 micromolar) and uncompetitive with respect to shikimate-3-P (K_i′ = 18.3 micromolar). All data were consistent with a mechanism of inhibition in which glyphosate competes with PEP for binding to an [enzyme:shikimate-3-P] complex and ultimately forms the dead-end complex of [enzyme:shikimate-3-P:glyphosate].     

3-Deoxy-D-arabino heptulosonate 7-phosphate synthase from Zea mays: General properties and regulation by tryptophan

In extracts from Zea mays shoots, the presence of thiol compoundsin the extraction buffer was necessary to get an active 3 deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP) synthase. Its pH optimumfor activity was about 7.5. Of the different cations tested,only Mn⁺⁺ was an activator. Enzyme stability was optimal inTris-HCl buffer, pH 7.5, that contained a reducing agent, Mn⁺⁺and a polyol. Contrary to other reports, phosphoenolpyruvate(PEP) did not stabilize the preparation significantly. The synthaseexhibited high affinities for both erythrose-4-phosphate (Km:0.24 mM) and PEP (Km: 0.31 mM). Its specific activity was highestin young shoots. Corn DAHP synthase was inhibited in vitro by tryptophan. Moreover,the enzyme was retarded on a tryptophan agarose affinity column,but it was removed with the bulk of protein from the same supportwhen eluted with buffer containing tryptophan. Inhibition whichwas easily lost during storage at 4°C was pH dependent andincreased during development. Maximal inhibition, about 60%with 1 mM tryptophan, was observed in extracts from 8 day-oldshoots. Phenylalanine and tyrosine were not inhibitory, andno synergistic effects were observed when the aromatic aminoacids were tested in combination. Isoenzymes could not be demonstrated. (Received April 23, 1980; )     

Selective Effect of Salt Stress on the Activity of Two ATPases in the Cell Membrane of Nostoc muscorum

ATPase activity in the cell membrane of a salt-stressed cyanobacterium,Nostoc muscorum M-14, was examined cytochemically by three differentstaining protocols. Application of Hulstaert's method resultedin distinct precipitation of the reaction products of ATPaseinside the cell membrane exclusively. No reaction products wereformed when ATP was replaced by GTP or when dicyclohexylcarbodiimideor N-ethylmaleimide was present in the reaction mixture. Bycontrast, low levels were detectable after the reaction in thepresence of ouabain. Bafilomycin did not affect the formationof products. Mayahara's method, which is considered to demonstratethe reaction of Na⁺,K⁺-ATPase activity, revealed the presenceof a ouabain-sensitive Na⁺,K⁺-ATPase in the cell membrane, whileWachstein-Meisel's method revealed the presence of an ATPaseactivity that was resistant to ouabain. It appears, therefore,that cell membranes of Nostoc muscorum contain both ouabain-sensitiveATPase and ouabain-insensitive ATPase. Comparison of the stainingprofiles of salt-stressed cells with those of control cellssuggested that a high-salt environment activates the ouabain-sensitiveNa⁺,K⁺-ATPase, which seems likely to be involved in the effluxof Na⁺ ions. (Received February 7, 1995; Accepted August 9, 1995)     

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L--aminooxy--phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10^-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10^-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a super-induction of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K _I=2.4·10^-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K _I=1.8·10^-4).Abbreviations PAL phenylalanine ammonia lyase - L-AOPP L--aminooxy--phenylpropionic acid     

Isoforms of NADP-dependent Malic Enzyme in Tissues of the Greening Maize Leaf

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40 [EC] ) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min^–1 mg^–1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min^–1 mg^–1 protein (3.2 ? 0.5 µmolmin^–1 mg^–1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min^–1 mg^–1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C₄, isozymes, malic enzyme, photodifferentiation, Zea mays     

Inhibition of Anthocyanin Synthesis by Cobaltous Ions in the First Internode of Sorghum bicolor L. Moench

Cobaltous ions (Co²⁺) inhibited light-mediated anthocyanin synthesisin excised first internodes of Sorghum bicolor (L.) Moench.Studies with precursors/intermediates of the anthocyanin biosyntheticpathway, such as acetate, phenylalanine, t-cinnamic acid, tyrosine,and p-coumaric acid were undertaken to identify the metabolicsite at which Co²⁺ ions inhibit anthocyanin synthesis. p-Coumaricacid partially restored anthocyanin synthesis. No other intermediatewas effective in bringing about recovery. It is suggested thatCo²⁺ might interfere with process/es which leads to the formationof p-coumaric acid, an intermediate of the anthocyanin biosyntheticpathway. Key words: Sorghum bicolor (L.) Moench, anthocyanin, cobaltous ions, phenylalanine ammonia-lyase     

Induction of Phenylalanine Ammonia-Lyase Activation and Isoflavone Glucoside Accumulation in Suspension-Cultured Cells of Red Bean, Vigna angularis, by Phytoalexin Elicitors, Vanadate, and Elevation of Medium pH

Treatment of suspension-cultured cells of red bean, Vigna angularis,with nigeran resulted in an accumulation of isoflavone glucosides,such as daidzein 7-O-ß-D-glucoside, daidzein 7,4'-di-O-ß-D-glucoside,and 2'-hydroxydaidzein 7,4'-di-O-ß-D-glucoside, whichwas accompanied by a transient increase in the activity of phenylalanineanimonia-lyase (PAL). Similar effects were also seen with otherphytoalexin elicitors, such as RNase A and cell wall componentsof Phytophthora megasperma var. sojae. Interestingly, the accumulation of isoflavone glucosides andthe transient increase in PAL activity were induced also byvanadate, a specific inhibitor of plasma membrane adenosinetriphosphatase. K₃PO₄ showed similar effects, but this was ascribedto the elevation of medium pH caused by adding this basic salt.In fact, merely raising the pH of the medium was found to besufficient for the induction of PAL activity. Experiments usinginhibitors showed that the induction depends on RNA and proteinsyntheses. The results are discussed in relation to the possiblemechanism of action of phytoalexin elicitors. ¹ Present address: Laboratory of Biochemistry, Faculty of Agriculture,Nagoya University, Furocho, Chikusa, Nagoya 464, Japan.     

Effects of Glyphosate on Isolated Maize Mitochondria

The effects of the herbicide glyphosate (K⁺ salt) on isolatedmaize mitochondria have been investigated. Protein synthesis,oxygen uptake (state 3 and state 4 respiration) and passiveswelling were inhibited at concentrations in the 10^–6–10^–2M range. No decrease of the respiratory control ratio (RCR)or stimulation of ATPase activity by glyphosate (K+ salt) wereobserved. It is concluded that the previously reported decreaseof the RCR and ATPase stimulation by glyphosate (isopropylaminesalt) were probably due to the isopropylamine moiety or to impuritiesof the technical product. Key words: Glyphosate, Herbicide action, Maize, Mitochondria     

L-Phenylalanine ammonia-lyase activity in asparagus spears: Effects of excision and incubation

Incubation of disks sectioned from the basal portion of asparagusspears resulted in a 5-fold increase in L-phenylalanine ammonia-lyase(PAL) (E.C. 4.3.1.5 [EC] ) activity over the level initially presentin the intact tissue. The enhanced activity developed rapidly,with only a slight lag, increasing to a maximum level at 30hr. Thereafter, the level of activity decreased to 50% of maximumactivity and appeared to have attained a new, higher steady-statelevel after 72 hr of incubation. Similar levels of activitydeveloped in basal disks incubated either in buffer solutionor in air, and light had no effect on enzyme activity. The excision-promoted increase in enzyme activity was preventedby cycloheximide (20 ppm) but, unlike some other tissues, delayedaddition of the antibiotic to incubating disks promoted lossof the lyase activity. The phenylpropanoid end products, transcinnamate,p-coumarate and ferulate (at 10^–3 M each), in decreasingorder of effectiveness, also inhibited the excision-promotedincrease in enzyme activity and caused a loss of the enhancedactivity in the incubated disks. The possibility is discussed that the activity initially presentin the spears is under separate control from that activity inducedby excision. (Received April 22, 1972; )     

Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H₂ oxidase. The dehydroquinate synthaserequired Co²⁺ and NAD as cofactors. Co²⁺ could be replaced byCu²⁺ at 0.1 mM, but Cu²⁺ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co²⁺ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN₃or NaCN. Heavy metal ions, such as Ag⁺ and Hg²⁺, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )     

Kinetics of Root Elongation of Maize in Response to Short-Term Exposure to NaCl and Elevated Calcium Concentration

To study the effect of salt (NaCl) on root elongation we developeda device that measures this effect by means of a Linear VariableDifferential Transformer (LVDT). To test the efficacy of thedevice we performed experiments demonstrating that (a) ratesof elongation of primary maize (Zea mays L.) roots were comparableto elongation rates of primary roots growing freely in solutionculture; and (b) chilling and low O₂ concentrations of the solutionelicited the expected responses. Inhibition of root elongation by 75 mol m^–3 NaCl was gradual.At an iso-osmotic concentration, mannitol did not inhibit rootgrowth, suggesting that the inhibition was not due to osmoticfactors but rather to effects of salt on metabolism. The additionof supplemental Ca (10 mol m^–3) ameliorated this stressfulcondition. Timing of the application of Ca was critical. Treatmentwith Ca after addition of NaCl only partially restored growth,but pretreatment with Ca completely prevented the inhibitionof growth by salt stress. Key words: Root growth, Zea mays L., salinity     

Coleoptile Senescence in Rice (Oryza sativa L.)

We investigated the cellular events associated with cell deathin the coleoptile of rice plants (Oryza sativa L.). Seeds germinatedunder submergence produced coleoptiles that were more elongatedthan those grown under aerobic conditions. Transfer of seedlingsto aerobic conditions was associated with coleoptile opening(i.e. splitting) due to death of specific cells in the sideof the organ. Another type of cell death occurred in the formationof lysigenous aerenchyma. Senescence of the coleoptile was alsonoted, during which discolouration of the chlorophyll and tissuebrowning were apparent. DNA fragmentation was observed by deoxynucleotidyltransferase-mediateddUTP nick end labelling (TUNEL) assay, and further confirmedby the appearance of oligonucleosomal DNA ladders in senescentcoleoptile cells. Two nucleases (Nuc-a and Nuc-b) were detectedby in-gel-assay from proteins isolated from coleoptiles. Nuc-a,commonly observed in three cell death phases required eitherCa²⁺or Mg²⁺, whereas Nuc-b which appeared during senescencerequired both Ca²⁺and Mg²⁺. Both nucleases were strongly inhibitedby Zn²⁺. Copyright 2000 Annals of Botany Company Aerenchyma, rice, cell death, coleoptile, fragmentation, nuclease, Oryza sativa, senescence, split, submergence, TUNEL     

Isolation and characterization of the cell wall receptor of 14C-malformin in Phaseolus vulgaris L.

¹⁴C-malformin attaches to at least two cell wall receptors inPhaseolus vulgaris. One receptor was extracted with Tris buffer(pH 8.5) and the other with 0.1 N NaOH. In both cases, priortreatment of the walls with wall degrading enzymes (macerase,cellulysin) was required. The two receptors differed with regardto ultrafiltration and gel filtration chromatography. The Tris-extractedreceptor is a protein, probably a glycoprotein, which containshydroxyproline and sulfhydryl groups. Although cuttings nottreated with malformin had Tris-extractable receptor, formationof the receptor appeared to be enhanced by malformin. ¹ Present address: American Cyanamid, P. O. Box 400, Princeton,New Jersey 08540, U. S. A. (Received August 2, 1976; )