14C-sucrose uptake by internode tissue

The transport and accumulation of ¹⁴C activity in decapitated, non-growing internodes of Phaseolus vulgaris L following application of ¹⁴C-sucrose or ¹⁴CO₂ is stimulated by application of auxins, cytokinins and gibberellins. The possibility that these hormone-directed effects may be mediated by stimulating the metabolism or storage of ¹⁴C-sucrose (i.e. by increasing sink demand) in the ground tissues of the stem was studied by investigations into the kinetics of ¹⁴C-sucrose uptake by thin slices of internode tissue of tomato and P. vulgaris. Sucrose uptake appears to be a carrier-mediated process, requiring active cell metabolism, and is shown to be stimulated by fusicoccin but not by indol-3yl-acetic acid (IAA). IAA only stimulates ¹⁴C-sucrose accumulation when long-distance transport is involved and it is suggested that IAA acts by stimulating the unloading of sucrose via an apoplastic pathway.     

Auxin transport: a new synthetic inhibitor

The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-¹⁴C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-¹⁴C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-¹⁴C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.     

Transport of the auxin, picloram, through petioles of bean and coleus and stem sections of pea

The transport of the synthetic auxin, picloram (4-amino-3,5,6-trichloropicolinic acid) was investigated in sections of petioles of Phaseolus vulgaris L. and Coleus blumei Benth. and stems of Pisum sativum L. Transport of ¹⁴C-picloram was basipolar in all tissues, although the degree of polarity was dependant on age. The velocity of picloram movement was calculated at between 0.75 and 1.11 mm/hr. The amount moved in a given time, the flux, was dependant on the concentration applied and the length of the sections used. Picloram did not appear to be metabolized by the tissues during the transport experiments. When compared to the movement of other growth regulators, picloram transport bears marked similarities to that of 2,4-dichlorophenoxyacetic acid.     

The Uptake and Fractional Distribution of Differentially Labeled Indoleacetic Acid in Light Grown Stems

The uptake of exogenously applied indoleacetic acid (IAA) by light grown stems of bean (Phaseolus vulgaris L. cv. Red kidney) and pea (Pisum sativum L. cv. Alaska) was examined. The IAA was labeled in the 1 and 2 side chain positions with ¹⁴C and the 5 ring position with ³H. The distribution of label in the sections was analyzed by recording the elution into water, ethanol and 1.0 N NaOH, and the amount in the insoluble residue also recorded. Total uptake consisted of a rapid uptake for about 1 h followed by continued uptake at a slower rate for 24 h to give a radioactive concentration in the tissues four to five times, that of the external solution. Most of the radioactivity was initially extractable by water, later by ethanol. With IAA-2-¹⁴C there was a slow increase in radioactivity in NaOH and residue fractions but with IAA-1-¹⁴C most of the radioactivity was present in insoluble residue at times longer than 3 h. From the different residue patterns estimates of the extent of decarboxylation of the IAA were made. The radioactivity in the tissues was largely IAA after 1 h and the content increased until 6 h but there after there was little further increase. The water fraction initially contained the most IAA but by 24 h most IAA was found in the NaOH fraction in bean and the ethanol fraction in pea. The NaOH fraction was the only fraction in which the IAA content continually increased.     

Correlative Inhibition of Lateral Bud Growth in Pisum sativum L. and Phaseolus vulgaris L.: Studies of the Role of Abscisic Acid

The possibility has been investigated that abscisic acid (ABA)might act as a correlative inhibitor of lateral bud growth inPisum sativum and Phaseolus vulgaris. Application of ABA insmall quantities (2µg) to axillary buds on decapitatedplants of P. sativum caused appreciable inhibition of theirgrowth, and induced a compensatory growth of the bud on an adjacentnode. Application of this same quantity of ABA to axillary budson decapitated plants of Phaseolus vulgaris was without effect,but a high concentration in lanolin (1 mg g^–1) did substantiallyreduce bud outgrowth. Endogenous ABA-like substances in Phaseolusvulgaris, detected by bioassay and electron capture g.l.c.,were present in similar concentrations in shoot tips, lateralbuds on intact plants and lateral buds on plants decapitated24 h earlier. The effects of applied ABA suggested that it might be involvedin the mechanism of correlative inhibition in Pisum sativum,but it was not possible to test this hypothesis by determiningendogenous ABA levels in axillary buds because of their smallsize. The evidence presented here suggests that ABA is not acorrelative inhibitor in Phaseolus vulgaris even though at highconcentration it can inhibit the growth of axillary buds.     

Cell length,light and14C-labelled indol-3yl-acetic acid transport inPisum satisum L. andPhaseolus vulgaris L

The putative auxin-transporting cells of the intact herbaceous dicotyledon are the young, differentiating vascular elements. The length of these cells was found to be considerably greater in dwarf (Meteor) than in tall (Alderman) varieties ofPisum sativum L., and to be greater in etiolated than in light-grown plants ofP. sativum cv Meteor andPhaseolus vulgaris L. cv Mexican Black. Under given light conditions during transport these large differences in cell length did not influence the shapes of the transport profiles or the velocity of transport of¹⁴C-labelled indol-3yl-acetic acid (IAA) applied to the apical bud. However, in both etiolated and light-grown bean and dwarf pea plants the velocity of transport in darkness was ca. 25% lower than that in light. Under the same conditions of transport velocities in bean were about twice those observed in the dwarf pea. Exposure to light during transport increased the rate of export of¹⁴C from the labelled shoot apex in green dwarf pea plants but not in etiolated plants. The light conditions to which the plants were exposed during growth and transport had little effect on the rates of uptake of IAA from the applied solutions. The results indicate that the velocity of auxin transport is independent of the frequency of cell-to-cell interfaces along the transport pathway and it is suggested that in intact plants auxin transport is entirely symplastic.     

The specificity of auxin transport in intact pea seedlings (Pisum sativum L.)

Summary When eight ¹⁴C-labelled auxin and non-auxin compounds were applied to the apical buds of intact dwarf pea seedlings (Pisum sativum L.), only [1-¹⁴C]indoleacetic acid ([¹⁴C]IAA) and -[1-¹⁴C] naphthaleneacetic acid ([¹⁴C]NAA) underwent appreciable basipetal transport during the first 24 h; over a longer period (72 h) considerable basipetal transport of the auxin [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([¹⁴C]2,4-D) also occurred, but at a very much lower velocity (ca. 1.4–2.2 mm·h^-1). The movement of 2,4-D possessed many of the characteristics of a typical auxin transport. During uptake and transport IAA and NAA were extensively metabolised to the corresponding aspartates, and to ethanol-insoluble/NaOH-soluble compounds; little metabolism of 2,4-D was observed. None of the non-auxin compounds applied (sorbose, sucrose, leucine, adenine and kinetin) underwent appreciable basipetal transport from the apical bud. All but sorbose were extensively metabolised by the apical tissues. Little metabolism of sorbose itself was detected.The results suggest that the long-distance basipetal auxin transport system from the apical bud of intact plants is specific for auxins; the specificity may result from the affinity of auxins for specific transport sites.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

The Fate of Exogenously Applied Indoleacetic Acid in Light Grown Stems

Elongating segments from light grown pea (Pisum sativum L. cv. Alaska) and bean (Pbaseolus vulgaris L. cv. Red kidney) stems were incubated in 10^-5M indoleacetic acid (IAA)-1-¹⁴C,and -5-³H in the light. Radoactive derivatives, extracted in water, ethanol or ether, and 1 N sodium hydroxide at three different times were chromatographed in three separate systems and the different metabolites identified by their labeling and chromatographic characteristics. Major metabolites included indoleacetyl aspartate, possibly indoleacetyl glucoside, hydroxymethyloxindole, and in bean a further major unidentified compund. Other compounds occurred in lesser amounts. Indole aldehyde was present only in very small quantities. IAA breakdown commenced between 1 and 6 h of incubation, following which IAA decreased and most metabolites increased, though IAA was still present after 24 h. Alkaline hydrolysates contained mainly IAA at a level which changed little between 6 and 24 h.     

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.     

Effect of 2,4-dichlorophenoxyacetic acid on nuclear division in stem segments ofPisum sativum L. cultivatedin vitro

We have investigated the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on the appearance of nuclear fragments, caused by direct nuclear division, as well as on mitotic activity in cultivated internodial stem segments ofPisum sativum L., cv. Bördi, during 180 d of cultivation. Direct nuclear fragmentation (dNF) was indicated by the shape and structure of the nucleus as well as by the occurrence of 1C- and 3C-values of DNA, investigated cytophotometrically. The dNF occurred during the whole cultivation period in segments treated by 2,4-D in concentrations from 4 to 32 mg 1^?1. In the presence of 2 mg 1^?1 of 2,4-D the dNF existed in the explants only up to 90 d. Mitotic activity was not observed in the 2,4-D-free control but occurred during the whole cultivation period when 2,4-D was added in concentrations from 2 to 16 mg l^?1. In the presence of 32 mg l^?1 of 2,4-D the level of mitotic activity was very low at the beginning and ceased after 60 d in culture.     

The role of auxin efflux carriers in the reversible loss of polar auxin transport in the pea (Pisum sativum L.) stem

Correlatively inhibited pea shoots (Pisum sativum L.) did not transport apically applied ¹⁴C-labelled indol-3yl-acetic acid ([¹⁴C]IAA), and polar IAA transport did not occur in internodal segments cut from these shoots. Polar transport in shoots and segments recovered within 24 h of removing the dominant shoot apex. Decapitation of growing shoots also resulted in the loss of polar transport in segments from internodes subtending the apex. This loss was prevented by apical applications of unlabelled IAA, or by low temperatures (approx. 2° C) after decapitation. Rates of net uptake of [¹⁴C]IAA by 2-mm segments cut from subordinate or decapitated shoots were the same as those in segments cut from dominant or growing shoots. In both cases net uptake was stimulated to the same extent by competing unlabelled IAA and by N-1-naphthylphthalamic acid. Uptake of the pH probe [¹⁴C]-5,5-dimethyloxazolidine-2,4-dione from unbuffered solutions was the same in segments from both types of shoot. Patterns of [¹⁴C]IAA metabolism in shoots in which polar transport had ceased were the same as those in shoots capable of polar transport. The reversible loss of polar IAA transport in these systems, therefore, was not the result of loss or inactivation of specific IAA efflux carriers, loss of ability of cells to maintain transmembrane pH gradients, or the result of a change in IAA metabolism. Furthermore, in tissues incapable of polar transport, no evidence was found for the occurrence of inhibitors of IAA uptake or efflux. Evidence is cited to support the possibility that the reversible loss of polar auxin transport is the result of a gradual randomization of effluxcarrier distribution in the plasma membrane following withdrawal of an apical auxin supply and that the recovery of polar transport involves reestablishment of effluxcarrier asymmetry under the influence of vectorial gradients in auxin concentration.Abbreviations DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid This work was supported by grant no. GR/D/08760 from the U.K. Science and Engineering Research Council. We thank Mrs. R.P. Bell for technical assistance.     

The indirect role of 2,4-D in the maintenance of apical dominance in decapitated sunflower seedlings (Helianthus annuus L.)

When sufficient 2,4-D to maintain apical dominance for at least 21d was applied to the cut stem interface of sunflower seedlings which had been decapitated in the epicotyl, it could not be detected in the vicinity of the inhibited axillary buds 7d after application. Rather the 2,4-D concentrated at the stump apex where it was associated with formation of meristematic tissue. The results indicate an indirect role for 2,4-D in the maintenance of apical dominance in this system, possibly involving the induced meristematic activity.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - [¹⁴C]2,4-D 2,4-dichlorophenoxy[2-¹⁴C]acetic acid - IAA indol-3-yl-acetic acid     

Investigations on fungicides.

Uninoculated roots of pea (Pisum sativum) and French bean (Phaseolus vulgaris) have been shown to exude a number of antifungal compounds when grown in a non-sterile aqueous aerated medium. These have been identified and their possible importance in relation to disease resistance is discussed.     

Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots

In 1933 Thimann and Skoog demonstrated exogenous auxin repressionof lateral bud outgrowth in decapitated shoots ofVicia faba. This evidence has given strong support for a role of auxinin apical dominance. Most, but not all, investigators have confirmedThimann and Skoog's results. In the present study, auxin treatmentswere carried out on ten different species or plant types, manyof which were treated with auxin in different forms, media andunder different light conditions. The Thimann–Skoog experimentdid work for most species (i.e. exogenous auxin did repressbud outgrowth) including thedgt tomato mutant which is knownto be insensitive to auxin in certain responses. Toxic auxinsymptoms were observed in some but not all species. The Thimann–Skoogexperiment did not work for greenhouse-grownColeus or forArabidopsis. Light was shown to reduce apical dominance inColeus andIpomoeanil . apical dominance; lateral bud outgrowth; axillary bud; auxin; IAA; decapitation; Vicia faba ; Ipomoea nil ; Pisum sativum ; Phaseolus vulgaris ; Lycopersion exculentum ; dgt ; Coleus blumei ; Arabidopsis thaliana ; Helianthus annuus ; Thimann–Skoog     

Further biological characteristics of galactoglucomannan oligosaccharides

The biological activity of cell wall-derived galactoglucomannan oligosaccharides (GGMOs) was dependent on their chemical structure. Galactosyl side chains linked to the glucomanno-core influenced their inhibition of elongation growth of pea (Pisum sativum L. cv. Tyrkys) stem segments induced by 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of the number of galactosyl side chains in GGMOs caused stimulation of the endogenous growth. Modification on the glucomanno-reducing end did not affect significantly the activity of these oligosaccharides. GGMOs inhibited also the elongation induced by indole-3-acetic acid (IAA) and gibberellic acid (GA₃). In the presence of IAA the elongation growth was inhibited to 20 – 35 % after 24 h of incubation depending on GGMOs concentrations (1 μM, 10 nM, 0.1 nM), similarly as in the presence of 2,4-D, which confirms the hypothesis of GGMOs antiauxin properties. The elongation induced by GA₃ was inhibited to 25 – 60 %, however, the time course of inhibition was different compared with IAA and 2,4-D. The highest inhibition was determined already after 6 h of incubation with a significant decrease after this time. The results indicated a competition between GGMOs and growth regulators.     

Growth of Avena coleoptiles and pH drop of protoplast suspensions induced by chlorinated indoleacetic acids

Several indoleacetic acids, substituted in the benzene ring, were compared in the Avena straight growth bioassay. 4-Chloroindoleacetic acid, a naturally occurring plant hormone, is one of the strongest hormones in this bioassay. With an optimum at 10^-6 mol l^-1, it is more active than indoleacetic acid, 2,4-dichlorphenoxyacetic acid and naphthaleneacetic acid. 5-Chloro- and 6-chloroindoleacetic acids are very strong auxins as well. Other derivatives tested have a lower activity. 5,7-Dichloro- and 5-hydroxyindoleacetic acids have very low auxin activity at 10^-4 mol l^-1 and may be anti-auxins. Some of the derivatives were compared for their effect on pH decline in stem protoplast suspensions of Helianthus annuus L. and Pisum sativum L. The change of pH occurs without a lag period or with only a very short one. Derivatives which are very active in the Avena straight growth assay cause a larger pH decline than indoleacetic acid, while inactive derivatives cause effectively no pH decline.Abbreviations IAA Indoleacetic acid - 4-Cl-IAA 4-chloroindoleacetic acid - 5,7-Cl₂-IAA etc 5,7-dichloroindoleacetic acid     

Some properties of pea enation mosaic virus isolated from field pea and broad bean plants in Bohemia

Pea enation mosaic virus (PEMV) was isolated from disea sed field pea (Pisum sativum L.ssp. arvense A.Gr.) and broad bean (Faba vulgaris Moench) plants grown as filed crops at Bohumilice in Bohemia. The virus proved to be pathogenic for the following plant species:Pisum sativum L. cv. Raman,Faba vulgaris Moench,Lens culinaris Med.,Vicia sativa L.,Lathyrus odoratus L.,Glycine soja L.,Phaseolus vulgaris L.,Chenopodium amaranticolor Coste andReyn,Nicotiana clevelandi Gray,Trifolium incarnatum L. The dilution end point of the isolate was higher in pea plants (10^?4) than in broad bean plants (10^?2). The thermal inactivation point was 65–68° and the longevityin vitro between 10 and 14 days. According to the host range, symptoms on pea plants and physical properties the virus isolate studied resembles some isolates described in the U.S.A. and represents a PEMV strain different from those reported so far in Czechoslovakia.     

Auxin-Growth Relationships in Maize Coleoptiles and Pea Internodes and Control by Auxin of the Tissue Sensitivity to Auxin

Growth of a zone of maize (Zea mays L.) coleoptiles and pea (Pisum sativum L.) internodes was greatly suppressed when the organ was decapitated or ringed at an upper position with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) mixed with lanolin. The transport of apically applied ³H-labeled indole-3-acetic acid (IAA) was similarly inhibited by NPA. The growth suppressed by NPA or decapitation was restored by the IAA mixed with lanolin and applied directly to the zone, and the maximal capacity to respond to IAA did not change after NPA treatment, although it declined slightly after decapitation. The growth rate at IAA saturation was greater than the rate in intact, nontreated plants. It was concluded that growth is limited and controlled by auxin supplied from the apical region. In maize coleoptiles the sensitivity to IAA increased more than 3 times when the auxin level was reduced over a few hours with NPA treatment. This result, together with our previous result that the maximal capacity to respond to IAA declines in pea internodes when the IAA level is enhanced for a few hours, indicates that the IAA concentration-response relationship is subject to relatively slow adaptive regulation by IAA itself. The spontaneous growth recovery observed in decapitated maize coleoptiles was prevented by an NPA ring placed at an upper position of the stump, supporting the view that recovery is due to regenerated auxin-producing activity. The sensitivity increase also appeared to participate in an early recovery phase, causing a growth rate greater than in intact plants.     

The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments

Summary In previous research here, 3,5-diiodo-4-hydroxybenzoic acid (DIHB) was shown to promote the elongation of roots of cress (Lepidium sativum) seedlings growing in light, and to inhibit the auxin-induced production of ethylene in this tissue. Although DIHB is a cofactor for the oxidation of indole-3-acetic acid (IAA) by horse-radish peroxidase, it inhibits the decarboxylation of [1-¹⁴C]IAA by segments excised from cress roots. The inhibition by DIHB of ethylene production by this tissue does not, therefore, arise from a reduction of IAA levels. These findings are discussed in relation to the effects of DIHB on cress root growth.Abbreviations IAA indole-3-acetic acid - DIHB 3,5-diiodo-4-hydroxybenzoic acid - DCP 2,4-dichlorophenol - 2,4-D 2,4-dichlorophenoxyacetic acid This study forms part of a research project to be submitted by M.L.R. for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).