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     

Patterns of Tracheary Differentiation in Lettuce Pith Explants: Positional Control and Temperature Effects

When lettuce pith explants were cultured for 14 d on a xylogenicmedium, tracheary elements differentiated in greatest numbersbetween 25 and 30 °C. Numbers were depressed at lower temperaturesby slower development and at higher temperatures by adverseprocesses. The data did not support previous suggestions ofa great stimulation of xylogenesis above 30 °C and of aspecial sensitivity to low temperatures. Tracheary elements differentiated in various spatial patterns:as clumps in the peripheral callus, as strands which extendedradially and longitudinally from some of these clumps, as individuallarge tracheids especially at the more extreme temperatures,and as short strands associated with nodules and roots thatformed at favourable temperatures. We suggest that indoleacetic acid (IAA) has various roles inthe positional control of these tracheary patterns: (1) IAAdestruction at the explant surface leads to concentration gradientsthat inhibit tracheary induction close to the surface; (2) IAAtransport from the source in the culture medium to sinks especiallyat the explant surface, coupled with autocatalytic flow facilitation,leads to canalization along pathways that become meristematicand then trachcary strands; (3) the IAA flux (and associatedproton flux) along these pathways tend to orient cortical microtubulesat right angles to the flow, by some mechanism as yet unknown,and hence to control the orientation of tracheary element elongationand secondary wall banding. These suggestions, supported bymorphometric studies of tracheary element dimensions and orientations,and by experiments with localized IAA application, lead to ageneral interpretation of the development of polarity in plants. IAA, Lactuca sativa, lettuce, pith explants, positional control, temperature effects, tracheary element differentiation     

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     

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     

Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures

The differentiation of sieve and tracheary elements was studied in callus culture of Daucus carota L., Syringa vulgaris L., Glycine max (L.) Merr., Helianthus annuus L., Hibiscus cannabinus L. and Pisum sativum L. By the lacmoid clearing technique it was found that development of the phloem commenced before that of the xylem. In not one of the calluses was differentiation of tracheary elements observed in the absence of sieve elements. The influence of indole-3-acetic acid (IAA) and sucrose was evaluated quantitatively in callus of Syringa, Daucus and Glycine. Low IAA levels resulted in the differentiation of sieve elements with no tracheary cells. High levels resulted in that of both phloem and xylem. IAA thus controlled the number of sieve and tracheary elements, increase in auxin concentration boosting the number of both cell types. Changes in sucrose concentration, while the IAA concentration was kept constant, did not have a specific effect on either sieve element differentiation, or on the ratio between phloem and xylem. Sucrose did, however, affect the quantity of callose deposited on the sieve plates, because increase in the sucrose concentration resulted in an increase in the amount of callose. It is proposed that phloem is formed in response to auxin, while xylem is formed in response to auxin together with some added factor which reaches it from the phloem.     

Interaction of auxin, cytokinin, and gibberellin on cell division and xylem differentiation in cultured explants of Jerusalem artichoke.

Dark-cultured explants of parenchymatous cells isolated fromJerusalem artichoke tubers were induced to divide and differentiateas tracheary elements on Murashige and Skoog medium containingdifferent combinations of plant growth-hormones such as auxin(IAA), cytokinin (zeatin), and gibberellin (GA₃). Addition ofauxin to the growth-medium induced after a short lag period,very rapid cell division which was followed by differentiationof some of the divided cells as tracheary elements. At the optimallevel of IAA (5.0 mg/liter), the percentage of tracheids differentiatedwith respect to the total number of cell population was 13.54.When the explants were cultured in the presence of both auxin(IAA 5.0 mg/liter) and one cytokinin (zeatin 0.1 mg/liter),not only a strong interaction on cell division and trachearyelement formation was observed but also an increase in the percentageof tracheids differentiated in relation to the total cell population.Auxin-gibberellin and auxin-gibberellin-cytokinin treatmentsalso produced interaction on cell division and cytodifferentiation.In explants treated with the three growth-hormones about 20%of the total cell population differentiated as tracheary elements.Further, all the hormonal treatments gave different patternsof cytodifferentiation which reflected meristematic patterns. ¹ This research was supported by a grant from C. N. R. Italy. (Received April 18, 1973; )     

The effect of auxin starvation on the growth of auxin-dependent tobacco cell culture: dynamics of auxin-binding activity and endogenous free IAA content

The growth of a cell strain derived from the stem pith of tobacco(Nicotiana tabacum L., cv. Virginia Bright Italia) was investigatedin subcultures grown at various levels of synthetic auxins.Both partial and complete auxin starvation resulted in a decreaseof the frequency of cell division. For these treatments theendogenous free indole-3-acetic acid content increased substantiallyat the commencement of the exponential growth phase. The possibilitythat the receptivity of the cells to auxin changed during thegrowth cycle was examined by measuring the activity of a membrane-boundauxin-binding site. In subcultures grown in a medium with anoptimal auxin concentration the maximum auxin-binding activitywas restricted to the end of the exponential growth phase. Inthe cells cultivated in partially or completely auxin deprivedmedia the auxin-binding activity increased to varying extents.These results probably reflect mechanisms controlling both theintracellular content of free auxin and the sensitivity of thecells to exogenous auxin supply (including auxin binding) withrespect to the cell division and/or growth Key words: Nicotiana tabacum L., plant cell culture, IAA, auxin-binding site, cell division     

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.     

Effect of indole-3-acetic acid (IAA) and sucrose on vessel size and density in isolated stem segments of oak (Quercus robur)

The effects of several concentrations of indole-3-acetic acid (IAA) and sucrose on xylogenic cambial activity and secondary xylem differentiation were investigated in isolated stem segments of Quercus robur L. supplied with liquid medium in aseptic conditions. After 5 weeks of culture auxin controlled cambial cell division and the number and size of vessel elements even without sugar in the medium. Sucrose modified these IAA effects, although little cambial activity occurred without auxin. The xylem increment correlated with changes of auxin concentration with the optimum at 28.5 μ M IAA. The formation of wide vessels was correlated with the optimal concentration of auxin. The frequency of vessel differentiation increased with auxin concentration. High concentrations of sucrose (0.24 M and 0.96 M ) reduced both the number of vessels and their diameter. The frequency of vessel formation was inhibited more than the vessel size by changes of sugar concentration. The vessels formed under low concentrations of IAA were circular in transverse section. With increase in IAA concentration the shape of the vessel cross-section changed to oval with the largest dimension in the radial direction.     

Inhibition of transdifferentiation into tracheary elements by polar auxin transport inhibitors through intracellular auxin depletion

Polar auxin transport is essential for the formation of continuous vascular strands in the plant body. To understand its mechanism, polar auxin transport inhibitors have often been used. However, the role of auxin in vascular differentiation at the unicellular level has remained elusive. Using a Zinnia elegans cell culture system, in which single mesophyll cells transdifferentiate into tracheary elements (TEs), we demonstrated that auxin transport inhibitors prevented TE differentiation and that high concentrations of 1-naphthaleneacetic acid (NAA) and IAA overcame the repression of TE differentiation. Measurements of NAA accumulation with 3H-labeled NAA in the presence or absence of 1-N-naphthylphthalamic acid (NPA) revealed enhanced NAA accumulation within the cell. In the NPA-treated cells, intracellular free NAA decreased, while its metabolites increased. Therefore, the polar auxin transport inhibitors may prevent auxin efflux and consequently promote NAA accumulation in Zinnia cells. The excess intracellular NAA may also activate NAA metabolism, resulting in a decrease in free NAA levels. This depletion of free NAA may prevent TE differentiation. The decreased auxin activity in NPA-treated cells was confirmed by the fact that the DR5 (a synthetic auxin-inducible promoter)-mediated expression of a reporter protein was suppressed in such cells. Gene expression analysis indicated that NPA suppressed TE differentiation at an early process of transdifferentiation into TEs. Based on these results, the inter-relationship between auxin and vascular cell development at a cellular level is discussed.     

Histological responses of isolated leaves to hormones applied across a transverse gradient

The responses of petiolar tissue of isolated leaves of Argyreianervosa to hormones were investigated by maintaining a transversegradient of auxin and auxin antagonist or auxin and gibberellin.The auxin used was ß-indolylbutyric acid (IBA) andthe auxin antagonist applied was maleic hydrazide. Gibberellicacid was also applied simultaneously with IBA. The experimentwas designed in such a way that while the solution of one hormonewas applied internally to the petiole with a capillary tube,the external surface of the petiole came in contact with thesolution of the other hormone. Wounding was caused in the pith by inserting a capillary tube.The division of the parenchymatous cells bordering the woundwas greater and cell dimension was less when auxin was appliedinternally but cell division was restricted and cell dimensionincreased when the auxin antagonist was applied at high concentrations.Root primordia and vascular tissues were formed in the pithwhen the concentration of auxin in the vicinity was greater.But these processes were blocked when the concentration of theauxin antagonist was greater in the neighborhood. The effectof auxin and gibberellin was synergistic in inducing these processes. (Received May 23, 1980; )     

Direct Evidence for Cytodifferentiation to Tracheary Elements without Intervening Mitosis in a Culture of Single Cells Isolated from the Mesophyll of Zinnia elegans

A serial observation of the process of tracheary element differentiation from single cells isolated from the mesophyll of Zinnia elegans L. cv. Canary bird provided the first direct evidence for the cytodifferentiation without intervening mitosis. Percentage of the tracheary elements formed without cell division was about 60% of total tracheary elements formed on the 4th day of culture. The number of tracheary elements formed without intervening mitosis was not reduced in the presence of colchicine at the concentrations blocking cell division. These facts clearly indicate that cell division is not a prerequisite for tracheary element differentiation in this system.     

Histological responses of isolated leaves to hormones applied across a transverse gradient

The responses of petiolar tissue of isolated leaves of Argyreianervosa to hormones were investigated by maintaining a transversegradient of auxin and auxin antagonist or auxin and gibberellin.The auxin used was ß-indolylbutyric acid (IBA) andthe auxin antagonist applied was maleic hydrazide. Gibberellicacid was also applied simultaneously with IBA. The experimentwas designed in such a way that while the solution of one hormonewas applied internally to the petiole with a capillary tube,the external surface of the petiole came in contact with thesolution of the other hormone. Wounding was caused in the pith by inserting a capillary tube.The division of the parenchymatous cells bordering the woundwas greater and cell dimension was less when auxin was appliedinternally but cell division was restricted and cell dimensionincreased when the auxin antagonist was applied at high concentrations.Root primordia and vascular tissues were formed in the pithwhen the concentration of auxin in the vicinity was greater.But these processes were blocked when the concentration of theauxin antagonist was greater in the neighborhood. The effectof auxin and gibberellin was synergistic in inducing these processes. (Received May 23, 1980; )     

A Requirement for DNA Synthesis during Auxin Induction of Cell Enlargement in Tobacco Pith Tissue

IAA (indoleacetic acid) is known to induce cell enlargement without cell division in tobacco pith explants grown on an agar medium without added cytokinin. The very long lag period before IAA (2 × 10^?5M) stimulates growth, about 3 days, can be useful to study the metabolic changes which lead to the promotion of growth. When the disks are transferred to a medium without IAA after 2 days or less of treatment with IAA, the IAA does not stimulate growth. Disks transferred after 3 days, subsequently show an auxin response, almost as great as those given IAA continuously. At 5 × 10^?4M, 5-fluorodeoxyuridine (FUDR), which inhibits DNA synthesis by blocking formation of thymidylate, completely suppresses the lAA-induced growth if it is added together with the IAA or 1 day later. When the FUDR is given 2 days after the IAA, there is a small increment of auxin-induced growth, and an even greater amount if added after 3 days. The period when exogenous auxin must be present to stimulate growth corresponds to the period of FUDR sensitivity. The FUDR inhibition is prevented by thymidine but not by uridine. Other inhibitors of DNA synthesis, hydroxyurea and fluorouracil, also inhibit auxin-induced growth. Thus DNA synthesis seems to be required for auxin induction of cell enlargement in tobacco pith explants. In contrast, FUDR does not inhibit auxin-induced growth in corn coleoptile and artichoke tuber sections.     

POLARITY OF TRACHEARY REGENERATION IN YOUNG INTERNODES OF COLEUS (LABIATAE)

After Jacobs’ recent discovery of almost absolute basipetal polarity of IAA-¹⁴C movement through young internodes of Coleus, tracheary regeneration around a wound in the stem was re-investigated to see if it showed parallel changes from the less strict polarity of IAA-¹⁴C described decades ago. As determined from either counts of “complete regenerated strands” or from finer details of regeneration, tracheary regeneration was very strongly polar. If leaves were present only below the wound, no regenerated strands developed unless there was a sizeable length of leafless stem remaining above the wound. If there were leaves below the wound as well as above it the amount of regeneration was usually reduced. The short strands of acropetally regenerating tracheary cells, previously interpreted as resulting from acropetal IAA movement, were observed in plants with leaves above but not below the wound, and were not seen in plants with leaves only below the wound. Hence, they are more likely to result from basipetally moving IAA. Isolated patches of tracheary regeneration were observed under several conditions. The wound interfered with development of the leaf directly above the wound.     

Inhibition of xylogenesis by morphactin in pith parenchyma explants of Lactuca

Pith parenchyma explants of Romaine lettuce (Lactuca salivaLinn. var. Roman?) incubated in the dark for 7 days at 25?Con a nutrient medium containing sucrose, IAA. and kinetin exhibitedextensive differentiation of tracheary elements. The additionof CFL to the medium strongly inhibited tracheary element formation.The lack of tracheary strand formation in the CFL-treated explantssuggests the inhibition of auxin transport. Conclusive evidencethat CFL influences the anatomy of differentiating xylem elementswas lacking. The addition of CFL to various combinations ofxylogenic media was not stimulatory to xylem element formationbeyond the differentiation response observed in the absenceof CFL. Unique patterns of tracheary element formation producedby cytokinin media containing IAA, 2,4-D, and NAA, respectively,were abolished by CFL. As indicated by counts of total trachearyelements formed per explant, the addition of cysteine to a CFL-containingmedium reversed the inhibitory effect of CFL. Tracheary strandformation was not re-established in the explants cultured onthe cysteine+CFL medium. Tracheary element formation was completelysuppressed by TIBA. Cysteine had a slight effect on the inhibitionof differentiation by TIBA. These observations suggest thatCFL inhibits some sulfhydryl- containing system involved eitherin the process of xylem differentiation or in some prerequisiterole necessary for the induction of tracheary element formation. (Received December 27, 1972; )     

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     

CELL DIVISION IN RELATION TO CYTODIFFERENTIATION IN CULTURED PEA ROOT SEGMENTS

One mm-thick segments cut 10–11 mm proximal to the root tip of germinating seeds of garden pea Pisum sativum were cultured in sterile nutrient medium containing auxin in the presence and absence of kinetin. In the absence of added cytokinin, pericyclic proliferation occurred, the cortical tissues showed no proliferation and were sloughed off, and a callus tissue of diploid cells was formed. In the presence of kinetin concentrations from 0.1–1.0 ppm cortical cells of the segments were induced to divide, beginning at the third day. From experiments with ³H-thymidine incorporation at different times of culture, from cytological squash preparations and from histological sections it was shown that the cortical cells stimulated to divide by cytokinin underwent DNA synthesis prior to division, were polyploid, and following cell division rapidly underwent cytodifferentiation at 5–7 days to form mature tracheary elements. At 10 days, when over 300,000 new cells had been formed per segment about 16% of these cells had formed tracheary elements. It was concluded that cytokinin, together with auxin, was essential for the initiation of DNA synthesis in the cortical cells, for their subsequent division, and finally for their specific cytodifferentiation.     

Early embryo development in Fucus distichus is auxin sensitive

Auxin and polar auxin transport have been implicated in controlling embryo development in land plants. The goal of these studies was to determine if auxin and auxin transport are also important during the earliest stages of development in embryos of the brown alga Fucus distichus. Indole-3-acetic acid (IAA) was identified in F. distichus embryos and mature tissues by gas chromatography-mass spectroscopy. F. distichus embryos accumulate [(3)H]IAA and an inhibitor of IAA efflux, naphthylphthalamic acid (NPA), elevates IAA accumulation, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. F. distichus embryos normally develop with a single unbranched rhizoid, but growth on IAA leads to formation of multiple rhizoids and growth on NPA leads to formation of embryos with branched rhizoids, at concentrations that are active in auxin accumulation assays. The effects of IAA and NPA are complete before 6 h after fertilization (AF), which is before rhizoid germination and cell division. The maximal effects of IAA and NPA are between 3.5 and 5 h AF and 4 and 5.5 h AF, respectively. Although, the location of the planes of cell division was significantly altered in NPA- and IAA-treated embryos, these abnormal divisions occurred after abnormal rhizoid initiation and branching was observed. The results of this study suggest that auxin acts in the formation of apical basal patterns in F. distichus embryo development.