Determination of Tracheary Element Differentiation in Lettuce Pith Explants

Transfer experiments with lettuce pith explants revealed thatprovision of inductive concentrations of both cytokinin andauxin in the culture medium for the first 3 and 5 d respectivelywas sufficient to cause determination of tracheary elementsas shown by their subsequent differentiation in substantialnumbers in explants removed from the inductive hormonal stimuli. Determination, differentiation, tissue culture, IAA, zeatin, tracheary elements, lettuce, Lactuca     

IAA Amino Acid Conjugates Induce Differentiation of Tracheary Strands in Lettuce Pith Explants

Each of four amino acid conjugates of IAA was able to replacethe IAA requirement for xylogenesis in lettuce pith explants,when supplied at concentrations ten to 100 times those optimalfor IAA. Tracheary development induced by these conjugates tendedto be slightly slower and less in amount than with IAA, andthe tracheary strands shorter and less regular. Responses differedsomewhat among the four conjugates: IAA-D, L-aspartate gavedevelopment most like that with free IAA, and IAA-D, L-phenylalanineoften yielded the weakest tracheary development, while responsesto IAA-L-alanine and IAA-glycine were intermediate. The resultsare interpreted in terms of the ‘bound’ IAA conjugatesdiffusing into the pith explants and becoming xylogenic onlyon hydrolysis to ‘free’ IAA. As tracheary strandformation is believed to result from IAA fluxes, it seems thatthe free IAA also moved through the discs, presumably towardsthe surfaces where it degrades rapidly. Tracheary strand formationin these explants can be compared with vascular strand formationin the normal shoot tip, where IAA conjugates (auxin ‘precursors’)move acropetally and are hydrolysed to free IAA especially inthe young leaf primordia, we suggest, yielding local sourcesof IAA which may contribute both to the phyllotactic spacingof primordia and, moving basipetally, to the definition of theauxin pathways that develop as procambial strands behind individualleaf primordia. Lactuca sativa, lettuce, IAA conjugates, tracheary element differentiation, pith explants, xylem strands     

Effects of Auxin Concentration on the Dimensions and Patterns of Tracheary Elements Differentiating in Pith Explants

The optimal concentration of IAA (0.03 mM) for tracheary elementdifferentiation in lettuce pith explants was about ten timesgreater than the optimal concentration for callus proliferation.Related to this, the mean volume per tracheary element increasedwith increasing IAA concentration, 18-fold between 0.001 mMand 0.3 mM IAA. At the highest concentrations, some pith cellsappeared to differentiate directly into tracheary elements,without cell division, resulting in especially large trachearyelements. Tracheary strands developed at intermediate concentrationsof IAA, and led to a small increase in the mean length/breadthratio of tracheary elements. For tracheary elements differentiating from stem cambial derivatives,a reassessment of previous studies indicates that increase inauxin concentration brings greater tracheary element size atconcentrations up to the 0.03 mM optimum. Above this optimum,however, further increase in auxin concentration brings progressivelysmaller tracheary elements, as the high auxin curtails enlargementof the differentiating cells. This contrasts with the pith explants,in which tracheary element size increases with IAA concentrationmost markedly above the optimum concentration. The interpretationof these relations requires an understanding of the effectsof auxin concentration on interacting quantities such as initialsize of cells, rate of enlargement, and rate of differentiation. Lactuca sativa, lettuce, IAA concentration, pith explants, tracheary element dimensions     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

Stimulatory and Inhibitory Effects of Sucrose Concentration on Xylogenesis in Lettuce Pith Explants; Possible Mediation by Ethylene Biosynthesis

Numbers of tracheary elements differentiating in lettuce pithexplants rose with increase in concentration of sucrose in themedium up to an optimal concentration of 0·2%, and fellwith further increase in concentration to about one-tenth maximalat 3% sucrose. Although a few tracheary elements formed withoutexogenous sucrose, a very low concentration of sucrose (0·001%)was sufficient to stimulate additional xylogenesis. Pretreatmentof explants with 3% sucrose caused a persisting inhibition ofxylogenesis, especially in tissue that had been near the siteof sucrose application (sandwich technique). The requirementfor adequate, but not inhibitory, concentrations of sucrosefor xylogenesis may underlie the development of xylem alongsidethe sucrose-rich phloem in normal apical morphogenesis. For callus growth the response to sucrose was different: theoptimal concentration was 3%, with a broad plateau from 1 to4% sucrose. Sucrose concentrations of 2 to 3%, used in manytissue culture media, are thus roughly optimal for callus growth,but ten times the optimum for xylogenesis in lettuce pith explants. It is surprising that 0·001% (0·03 mM) sucrose,applied exogenously, can stimulate xylogenesis: endogenous sugarconcentrations are normally higher. Perhaps the stimulationis mediated by ethylene biosynthesis, which is known to be xylogenic.Rates of ethylene production per explant rose with increasingsucrose concentration from about 0·1 nl h^-1 at 0% sucroseto a slightly (significantly) higher level at 0·004%sucrose and to about 0·5 nl h^-1 at 3% sucrose. D -glucoseresembled sucrose in its effects on xylogenesis and ethyleneproduction, but L-glucose yielded no xylogenesis and littlestimulation of ethylene biosynthesis.Copyright 1994, 1999 AcademicPress Lactuca sativa, Coleus blumei, Nicotiana tabacum, lettuce pith explants, tracheary element differentiation, sucrose, glucose, ethylene     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

Exogenous Methionine as a Nutrient Supplement for the Induction of Xylogenesis in Lettuce Pith Explants

The induction of xylogenesis in explants of lettuce pith parenchymawas greatly influenced by the presence of exogenous methionine(0·025 to 0·05 µM) in the culture medium.At the various concentrations of methionine tested, trachearyelement differentiation was stimulated in the majority of theexplants. Differentiation, however, was markedly depressed ina small number of explants grown under the same cultural conditionsin the presence of methionine. Cytodifferentiation in controlexplants, cultured on a similar medium lacking methionine, gaveconsistent tracheary cell counts with little variation. Thesedata are consistent with the hypothesis that theenhanced productionof ethylene, due to the presence of methionine as a substrate,plays a role in the initiation of xylem differentiation. Theconcentration of ethylene in the cultured tissue may be a criticalfactor in determining whether the hormone will stimulate orsuppress the initiation of cytodifferentiation. Some thick-walledand pitted cells were observed, and these may represent partially-differentiatedxylem elements.     

The Role of Benzyladenine in the Differentiation of Tracheary Elements in Jerusalem Artichoke Tuber Explants Cultured in vitro

The role of benzyladenine (BA) in the differentiation of trachearyelements in Jerusalem artichoke (Helianthus tuberosus L.) tuberexplants was studied. For maximum differentiation of trachearyelements (25–30% of the cell population), treatment withoptimal concentrations of benzyladenine (5.0 mg dm^–3)in the presence of -naphthaleneacetic acid |NAA| (1.0 mg dm^–3)for the first 6 d was as effective as its continued presenceduring the entire 14 d period of study. A majority of the differentiatedtracheary element appeared between the 10th and 14th days ofculture. It was further observed that concentrations of activecytokinins in the tissue were considerably reduced within 2d after transfer from the BA-containing medium to a BA-freemedium. This was shown in three different ways: (1) monitoringthe amount of ethanol-soluble radioactivity at various timesafter transfer from |14C|-BA containing medium to BA-free medium;(2) bioassay of various cytokinin fractions from tissue extractseparated by thin layer chromatography; (3) indirect assay oftissue cytokinin activity through its interaction with abscisicacid for the promotion of auxin-induced cell division in thistissue. Both gibberellic acid (5.0 mg dm^–3) and abscisic acid(2–0 mg dm^–3) effectively inhibited the differentiationof tracheary elements even if provided after 6 d of pre-incubationin a high tracheid inducing medium. However, the appearanceof differentiated cells for the first 2 d after transfer wasnot significantly affected. A hypothetical scheme for the role of benzyladenine in the differentiationof tracheary elements in this tissue is discussed. It is suggestedthat during one or more critical cell divisions in the presenceof optimal levels of benzyladenine, a proportion of cells areinduced or committed for later differentiation into trachearyelements. The high concentrations of benzyladenine requiredduring induction are not needed during the intervening celldivisions, nor for the actual differentiation of the trachearyelements. Key words: Tracheary element differentiation, Jerusalem artichoke (Heliantlus tuberosus), Benzyladenine, Gibberellic acid, Abscisic acid     

Loss of Tonoplast Integrity Programmed in Tracheary Element Differentiation

A tracheary element (TE) is a typical example of a cell type that undergoes programmed cell death in the developmental processes of vascular plants. The loss of the selective permeability of the tonoplast, which corresponds to tonoplast disintegration, occurred after the cells commenced secondary wall thickening and played a pivotal role in the programmed cell death of TEs in a zinnia (Zinnia elegans L.) cell culture. A search for events specifically associated with the TE vacuole provided an important clue to the understanding of the cell death mechanism. The transport of fluorescein, a fluorescent organic anion, across the tonoplast declined drastically in differentiating TEs. The capacity of the vacuole to accumulate the probe was also impaired. Treatment with probenecid, an inhibitor of organic anion transport, caused rapid cell death of TEs and led to the ultimate disruption of the vacuole even in other types of cultured cells. These changes in vacuolar properties during TE development were suppressed by cycloheximide. Specific mRNA accumulation in cells cultured in a TE differentiation-inductive condition was abolished by probenecid. These results suggest that a change in vacuolar membrane permeability promotes programmed cell death in TEs.     

Isoperoxidase Patterns in Tobacco Pith and their Alteration Following Tissue Excision

Previous work has shown that young tobacco pith cells containlow peroxidase activity resolvable into two anodic iso-zymesby electrophoresis at pH 8.3 on starch gel. During normal cellenlargement and ageing in situ, these two isoenzymes increaseslowly in activity. By contrast, within 24 h after excisionof pith, high activities appear in several apparently new anodicand cathodic isoperoxidases. This development of new isoperoxidasesis prevented by physiological levels of IAA and by inhibitorsof RNA and protein synthesis. The present work describes additional quantitative isoperoxidasegradients, both vertical and horizontal, in the pith. Severaladditional isoenzymes, some of which are excreted into the mediumduring incubation, have been detected by improved electrophoretictechniques. Enzyme leakage can be prevented by incubation inmoist chambers and by auxin application through terminally placedagar blocks. With such techniques, new peroxidases form preferentiallynear cut surfaces within 12 h under aerobic conditions. Tissuesperipheral to the pith retard the development of new peroxidaseactivity. IAA in agar blocks applied terminally to a cut pithcylinder retards peroxidase formation mainly at the base. Theregulation of the quantity and quality of isoperoxidases seemsto be correlated with certain morphogenetic phenomena.     

The Role of Basipetal Auxin Transport in the Positional Control of Abscission Sites Induced in Impatiens sultani Stem Explants

If segments of Impatiens sultani stem are explanted and incubated,separation layers often form across them and lead to abscission.To test the suggested role of auxin concentration in controllingthe position of abscission sites, explants were labelled byapplying [¹⁴C]IAA to the shoot tip 4 h prior to explanting;transport of auxin applied in this way seems to resemble thatof endogenous auxin. During subsequent incubation of explantsfor 20 h, basipetal transport resulted in ¹⁴C accumulating justabove the base of the explants (nearly 80 % in the bottom 4mm of 24 mm explants). In internodal explants that had beenwounded at explanting by incising one side so as to sever avascular bundle, and in nodal explants with the leaf removed,the ¹⁴C also accumulated just above the wound or node to abouttwice the concentration otherwise expected; this accumulationwas probably due to basipetal transport being impeded by vasculardiscontinuity at the wound or node. Accumulation just abovethe base, or above a wound or node, resulted in gradients of¹⁴C concentration (presumably reflecting endogenous auxin concentration)decreasing in the morphologically upward direction at each ofthese three positions where abscission sites tend to occur. Impatiens sultani, abscission, auxin, IAA, node, polarized transport, positional control, separation layer, wounding     

Ontogeny and Hormonal Control of Polyphenoloxidase Isozymes in Tobacco Pith

Isozymes of tobacco pith polyphenoloxidases (o-diphenol oxidase, EC 1.10.3.1) were separated electrophoretically from fresh pith of intact plants and from cultured pith sections. Extracts of fresh pith contained a poorly resolved complex of two to three anodic bands after starch gel electrophoresis at alkaline pH. This anodic complex was more active with chlorogenic acid than with 3,4-dihydroxyphenylalanine and was found in greater activity per gram fresh weight of tissue in younger internodes than in older ones. The longitudinal gradient of activity was thus the opposite of that found for the constitutive isozymes of peroxidase.A well defined cathodic band of polyphenoloxidase activity appeared after culture of pith in modified White's medium with shaking. This band, which was more active with 3,4-dihydroxyphenylalanine than with chlorogenic acid, could be detected after 1 to 2 days of incubation. Its appearance was enhanced by the addition of 10 mum indoleacetic acid; kinetin (1 mum tended to prevent this indoleacetic acid effect). Such hormonal control is opposite to that previously reported for the rapidly appearing new isozymes of peroxidase.The pattern of the major isozymes associated with polyphenoloxidase activities differs from that of peroxidase.     

Lignification and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: A Role for Endogenous Ethylene

Lignification and xylogenesis were studied in lettuce (Lactucasativa L. cv. Romaine) pith parenchyma explants cultured ona Murashige and Skoog basal medium supplemented with indole-3-aceticacid, kinetin, and glucose. Lignin formation was observed tooccur in two distinct phases, one preceding xylem differentiation(days 0–3 of culture) and another coincident with maximalxylogenesis (days 4–7). The rate of soluble phenolic productionby these explants increased concomitant with the first phaseof lignification, then decreased during the second phase. Addition of silver, an ethylene antagonist, to the culture mediuminhibited the second phase of lignification and markedly reducedwall-bound peroxidase activity. Exogenous L-methionine, an ethyleneprecursor, completely reversed the inhibitory effect of silveron ligm6cation and wall-bound peroxidase activity. Silver increasedphenylalanine ammonia-lyase activity, but had no effect on solublephenolic production or soluble pcroxidase activity. These resultssuggest that ethylene may play a role in controlling lignificationduring xylogenesis by inducing wall-bound peroxidase activity. Key words: Auxin, Cytokinin, Ethylene, Xylogenesis, Lignification, Phenylalanine ammonia-lyase, Peroxidase     

Regulation of Tracheary Element Differentiation by Exogenous L-Methionine in Callus of Soya Bean Cultivars

The relationship between the induction of tracheary elementdifferentiation and exogenous L-methionine was examined in agar-growncultures of soya bean callus initiated from Glycine max L. ‘Wayne’and ‘Clark 63’. Although Wayne is a normal cultivarsoya bean, seedlings of Clark 63 exhibit abnormal growth at25 °C due to exessive ethylene biosynthesis at this temperature.Wayne callus showed increased xylogenesis in the presence ofexogenous L-methionine (3.7 µg 1^–1) in comparisonto IAA–KN controls at both 20 and 25 °C. Clark 63callus produced greater numbers of tracheary elements in responseto exogenous L-methionine only at 25 °C. The induction ofxylem differentiation was independent of the maintenance temperatureof the stock cultures of both cultivars. Xylogenesis initiatedbyan IAA–KN medium was inhibited by the addition of AgNO₃(20 mg 1^–1) to the extent of 76.5 per cent in cv. Wayneand 6 per cent in cv. Clark 63. The inhibitory effect was partiallyreversed by the addition of L-methionine (3.7 µg 1^–1)to the IAA–KN–AgNO₂ medium. These data support thehypothesis that xylogenesis in vitro involves auxin, cytokininand ethylene. differentiation, xylogenesis, L-methionine, ethylene, Glycine max L., soya bean, callus culture, auxin, kinetin     

Effects of Temperature and Various Red or Far-red Irradiations on Phytochrome- and Gibberellin A3-mediated Germination Control in Partially Hydrated Lettuce Seeds

Dark reversion of phytochrome in partially hydrated lettuceseeds (Lactuca sativa cv. Grand Rapids) is temperature dependent.After initial red irradiation (R) the higher the storage temperature,the higher the dark reversion rate. Following dark moist storage(DMS) at 30 ?C for 15 d none of the seeds receiving initialR germinated, whereas seeds stored at 0 ?C germinated nearlyas well (about 80%) as unstored controls. The half-time fordark reversion at 20 ?C and 30 ?C is 9 d and 3 d respectively.Repeated R treatments given at 5 d intervals during DMS at 20?C and 30 ?C maintained a high germination capacity. With threeor more R treatments the effect of high temperature largelydisappeared. Dark reversion of phytochrome was not observed in partiallyhydrated lettuce seeds receiving continuous red irradiation(cont R) for two or more days. The promotive effect of contR could be reversed at any time with a brief far-red irradiation(FR), indicating that the phytochrome system remained fullyphototransformable. With continuous far-red light (cont FR)the ability of gibberellin A₃ (GA₃) to stimulate germinationdisappeared and response to GA₃ also diminished in cont R followedby FR but at a slower rate indicating the induction of secondarydormancy in these partially hydrated seeds. This induction ofdormancy was retarded by repetitive or cont R but was enhancedby cont FR. The results of this study suggest a role for theaccumulated stable intermediates of phytochrome transformationin partially hydrated seeds with repeated or continuous R treatmentsand different effects of GA₃ and R in the regulation of germination. Key words: Phytochrome, Lactuca sativa, Seed germination, Temperature, Dark reversion of phytochrome, Seed water content     

Effects of Nutrient Limitation and {gamma}-Irradiation on Tracheary Element Differentiation and Cell Division in Single Mesophyll Cells of Zinnia elegans

The effects of nutrient limitation and -irradiation on trachearyelement differentiation and cell division were investigatedusing single cells isolated from the mesophyll of Zinnia elegans.When the phosphate concentration of the medium was reduced to10 µM (1/50 of Fukuda and Komamine's medium, 1980a), thefrequency of cell division during 4 days of culture decreased,while the frequency of tracheary element differentiation wasunaffected. -Irradiation with a dose of 92 Gy at 36 h of culturepreferentially and thoroughly suppressed cell division withoutreducing the number of tracheary elements formed. The appearanceof secondary cell wall thickenings was delayed by irradiation,but synchrony was maintained. Thus the Zinnia system previouslyreported [Fukuda and Komamine (1980a) Plant Physiol. 65: 57]was improved to give a more useful system for the study of cytodifferentiation,in which tracheary element formation occurred from single cellswithout cell division. ¹Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted February 22, 1986)     

Characteristics of the Inhibition of Induced Tracheary Element Differentiation by 3-Aminobenzamide and Related Compounds

3-Aminobenzamide (3-ABAM), a specific inhibitor of the nuclearenzyme ADP-ribosyl transferase (ADP-RT) has been previouslyshown to inhibit the differentiation of tracheary elements withoutaffecting cell division (Hawkins and Phillips, 1983). This effectis confirmed here in cultured explants of pea as well as Jerusalemartichoke, and total inhibition of direct differentiation incultures of gamma-irradiated immature artichoke tubers demonstrated.In studies on the timing of action of 3-ABAM, cells were foundto be sensitive only during the 6 h period prior to the onsetof visible differentiation, i.e., after the final mitosis whencells were known to be in the G₁/G₀ phase of the cell cycle.The duration of effectiveness of 3-ABAM was limited to a maximumof 3–4 d, apparently due to metabolic degradation of theinhibitor. Other reported ADP-RT inhibitors and their analogueswere found to be toxic in this system. The blocking of differentiationby 3-ABAM was freely and rapidly reversed on removal, and bythis means the maturation time for differentiating trachearyelements was estimated at 6 h. The potential of studies on ADP-RTactivity, and its possible role in regulating DNA strand breakageand repair for advancing our understanding of the molecularmechanisms involved in xylem differentiation are discussed. Key words: Tracheary differentiation, 3-Aminobenzamide, Tissue culture     

Ethylene Biosynthesis and Xylogenesis in Lactuca Pith Explants Cultured In Vitro in the Presence of Auxin and Cytokinin: The Effect of Ethylene Precursors and Inhibitors

The possible involvement of ethylene in the induction of xylemdifferentiation was studied in lettuce (Lactuca saliva L. cv.Romaine) pith parenchyma explants. The addition of the ethyleneprecursors L-methionine (0.25 µM), S-adenosylmethionine(25 µM) and 1-aminocyclopropane-l-carboxylic acid (0.01µM), or the ethylene-releasing agent 2-chloroethylphosphonicacid (1.0 µM), to a standard IAA-kinetin-containing mediumenhanced xylogenesis compared to control explants cultured inthe absence of these compounds. In the presence of the ethyleneinhibitors aminoethoxyvinylglycine, Co(NO₃)₂ and AgNO₃, xylogenesiswas inhibited. Inhibition of xylogenesis by aminoethoxyvinylglycine(75 µM), Co(NO₃)₂ (50 µM) and AgNO₃ (6.0 µM)was reversed by exogenous 1-aminocyclopropane-l-carboxylic acid(0.01 µM), 2-chloroethylphosphonic acid (5.0 µM)and L-methionine (0.25 µM), respectively. Ethylene productionby explants cultured on media containing L-methionine or 1-aminocyclopropane-l-carboxylicacid was greater than the biosynthesis of ethylene by explantscultured in the absence of these compounds. The incorporationof 2-chloroethylphosphonic acid into the culture medium resultedin higher rates of ethylene production compared to explantscultured on the IAA-kinetin medium. The presence of either aminoethoxyvinylglycineor Co(NO₃)₂ inhibited ethylene production by explants culturedon the IAA-kinetin medium. The data support the hypothesis thatethylene plays a positive role in the initiation of xylem differentiation. Key words: Xylogenesis, Differentiation, Ethylene, IAA, Kinetin, Lactuca sativa     

Induced Abscission Sites in Internodal Explants of Impatiens sultani: A New System for Studying Positional Control: with an Appendix: A Mathematical Model for Abscission Sites

Intemodes from Impatiens sultani shoots, explanted into sterileculture, often developed a transverse separation layer afterone to two weeks and the top then abscised from the bottom ofthe explant. Such abscission occurred more rapidly and in agreater proportion of explants when 00001 per cent auxin (IAA)was provided basally and when younger intemodes and shorterexplants were used. The distance of the separation layer fromthe base of the explant varied little with explant length, butincreased with the concentration of auxin applied basally. It seems that in this adventitious abscission the processesof positional definition and differentiation proceed withoutpause, whereas in normal abscission the position is definedearly in development but the final stage of differentiationof the separation layer is delayed until much later when theorgan senesces. To account for the results from the internodal explants andfrom surgical operations on shoots as well as for the characteristicposition of abscission sites of leaves and fruits, we suggestthat the position of abscission is controlled primarily by auxinacting as a morphogen: abscission sites occur at Y-junctionsjust above the base of the arm with the lower activity and auxinstatus, or in single axes above a region of higher auxin status.In both sites, the auxin concentration decreases in the apicaldirection. This hypothesis is supported by a mathematical model (see Appendix)of the interaction of diffusive and polar transport in controllingthe concentration gradient along intemodes with specified auxinconcentrations maintained basally. The model allows predictionsconcerning the site and timing of abscission which accord withobservations on intemodal explants. Impatiens sultani Hook., abscission, auxin, differentiation, diffusion coefficient, IAA, morphogen, polar transport coefficient, positional control, separation layer     

In Situ Detection of nDNA Fragmentation during the Differentiation of Tracheary Elements in Higher Plants

Programmed cell death (pcd) is thought to occur during the autolysis of xylem vessels. Although several ultrastructural aspects of this differentiation process have been characterized, certain key aspects of this process remain unsolved. Here we demonstrate in pea (Pisum sativum) that nuclei of vessel elements undergoing pcd contain fragmented nDNA. This finding may provide evidence for the activation of a DNA degradation mechanism prior to the final disruption of the nucleus that occurs during the autolysis stage of this differentiation process. In situ detection of DNA fragmentation in nuclei of vessel elements undergoing pcd may therefore suggest that this death process involves the activation of a mechanism for DNA degradation, similar to that activated during apoptosis in animal cells. In addition, this differentiation process may serve as a useful positive control for the in situ detection of pcd in other developmental pathways and during the hypersensitive response of plants to avirulent pathogens.