Studies on the movement of indole auxins in willow (Salix viminalis L.)

Summary Auxin activity was detected in honeydew obtained from the aphid Tuberolachnus salignus (Gmelin) feeding on willow (Salix viminalis). Active uptake of ¹⁴C-indolyl-3-acetic acid (IAA) into the sieve tubes was demonstrated by irrigating the cambial surface of willow bark with ¹⁴C-IAA solution and assaying aphid stylet exudate. When, however, ¹⁴C-IAA was applied to the peridermal tissues of the bark or to a mature leaf most of the radioactivity (collected in honeydew or stylet exudate) co-chromatographed with indolyl-3-acetyl-aspartic acid (IAAsp). The presence of IAAsp in honeydew was not affected by extraction procedure or by aphid metabolism. Honeydew obtained from willow treated with ¹⁴C-tryptophan contained only ¹⁴C-tryptophan. When ¹⁴C-IAA was applied in agar to the cut end of willow segments the radioactivity was found to move in a basipetally polar manner. The direction of movement of radioactivity in the sieve tubes, however, was found to be influenced by the proximity of the roots. Nevertheless, there was evidence that endogenous auxin in the sieve tubes does move in a predominantly basipetal direction.     

Movement of Water and Solutes in Sieve Tubes of Willow in Response to Puncture by Aphid Stylets. Evidence against a mass flow of solution

Experiments are described in which bark strips of willow were sealed to polythene tubes having two compartments. This allowed investigations to be made of the transport along the sieve tubes of tritiated water, ¹⁴C-labelled sugars, and ³²P-phosphates from one compartment, towards a stylet situated in the bark over the other compartment. Although activity from both ¹⁴C and ³²p was detected in the stylet exudate usually within 1 hour from isotope application, tritium activity was never detected even after a period of 8 hours in most experiments, though in certain cases, very low activities were detected after 4 hours. Subsequent experiments in which stylets were sited over both compartments showed that tritium activity moved laterally into the punctured sieve element more rapidly than either ¹⁴C or ³²P. Experiments using both live and dead bark in which stylets were not employed, showed that within 4 hours tritium activity had moved by diffusion along the whole length of a bark strip, therefore after this time tritium activity could have moved into the stylet exudate by a diffusional process. The lack of rapid longitudinal movement of tritiated water along the sieve tubes, indicates that the transport process is unlikely to be a mass flow of solution.     

Patterns of translocation and metabolism of 14C-labelled IAA in the phloem of Willow

Summary When 2-¹⁴-C-labelled IAA was applied to an isolated segment of Willow via a bark abrasion the pathway of transport of this compound was found to be located in the sieve elements as evidenced by the pattern of activity found in honeydew excreted by individuals of Tuberolachnus salignus (Gmelin) feeding on the segment.Further experiments have established that polarity of transport of ¹⁴C-IAA occurs in a basipetal direction when isolated segments of willow are orientated in a vertical position, with the morphological apex uppermost. No polarity was found when segments were orientated in a vertical position with the morphological base uppermost, or when the segments were orientated in a horizontal position. The metabolism of ¹⁴C-IAA was also studied with respect to orientation. It was shown that the conversion rate of IAA to IAA aspartate was influenced by the orientation of the segment. It is considered that this is not a direct effect of orientation on the rate of metabolism of IAA.     

Pathways of auxin transport in the intact pea seedling (Pisum sativum L.)

Summary When small colonies of the pea aphid [Acyrthosiphon pisum (Harris)] were established on the stem of Meteor Dwarf Pea seedlings (Pisum sativum L.), ¹⁴C was found in the honeydew 4.5 h after applying IAA-1-¹⁴C to a fully-expanded foliage leaf. In contrast, no activity was found in the honeydew or aphids 4.5 h after the application of IAA-1-¹⁴C to the intact apical bud even though the internode upon which the aphids were feeding contained high levels of ¹⁴C. The lack of radio-activity in aphids feeding on stems to which IAA-1-¹⁴C was applied via the apical bud was found not to be influenced by the internode position or by the transport interval allowed (up to 24 h).Radioactivity derived from either foliar or apical applications of IAA-1-¹⁴C was not transported through stem tissues killed by heat treatment. Xylem function was shown not to be impared by the heat treatment employed.It was concluded that the long-distance transport of IAA from the apical bud of intact pea seedlings does not take place in the phloem sieve tubes involved in the transport of metabolites from foliage leaves, or in the non-living tissues of the xylem.     

Distribution of growth regulators and sugars by the tangential and radial transport systems of stem segments of Willow

Summary The tangential transport of labelled growth regulators and sugars, through the bark of isolated stem segments of Willow (Salix viminalis L.), has been studied. It has been shown that these compounds have distinct patterns of distribution, particularly in the case of plant growth regulators. IAA moves preferentially into the lower half of a horizontal stem, kinetin into the upper half when these substances are applied via a bark abrasion made on the side of the stem. Distribution patterns of sugars were more variable, in some instances upward movement occurred, in others downward. The distinctness of the distribution patterns was accentuated by pretreatment of the stem segments in a horizontal position for 7 days prior to experimentation. Radial movement of labelled IAA and kinetin, centrifugally from the centre of the xylem to the bark, showed no difference in distribution between the upper and lower portions of the stem.     

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     

Interaction of Kinetin and Indoleacetic Acid in the Avena Straight-Growth Test

Kinetin has a stimulating effect in the Avena straight-growth test. The action of different concentrations of kinetin, 2.5 × 10^?7, 2.5 × 10^?6 and 2.5 × 10^?5M, in combination with different concentrations of IAA was studied in this test. It was shown that the effect of low IAA concentrations, 0.25 × 10^?7 and 1 × 10^?7M, was strongly enhanced by the addition of all the kinetin concentrations investigated. The effect of the highest IAA concentrations, 25 × 10^?7 and 100 × 10^?7M, on the other hand, was inhibited relatively strongly by the highest employed concentration of kinetin. The results are explained as due to a kinetin-produced increase of auxin in the coleoptile segment, which in combination with low IAA concentrations can lead to a growth stimulation and with high IAA concentrations to a growth inhibition. Since kinetin in purification and chromatography of auxin can partly follow IAA, thereby affecting the quantitative yield, it is emphasized that, prior to the test, auxin extracts containing cytokinins should be freed from the latter by, for example, gel filtration or paper electrophoresis.     

The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 μg/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 μg/mL and 20 μg/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 μg/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.     

The Movement of Sugars into the Sieve Elements of Bark Strips of Willow: II. EVIDENCE FOR TWO PATHWAYS FROM THE BATHING SOLUTION

It is suggested that when ¹⁴C-labelled sucrose, or its constituenthexoses, are applied to the cambial surface of a bark strip,the sugars can move into the sieve elements by two pathways.The first is a direct one which probably involves the companioncells; in it the labelled sugars do not mix with pools of unlabelledsugars before entry into the sieve elements. Entry by this pathwayleads to activity in a wide range of compounds in the sieve-tubeexudate. The second pathway is an indirect one, involving thestorage parenchyma of the phloem, where considerable metabolicchanges take place involving the sugar applied to the strip,but the products of these changes, apart from sucrose, are unableto move into the sieve elements. These results are discussed in relation to published work onthe cytology of angiosperm phloem, and the results of otherinvestigators on the movement of sugars into plant cells.     

Estimation of the volumetric elastic modulus and membrane hydraulic conductivity of willow sieve tubes

Severed aphid stylets were used to follow the kinetics of sieve tube turgor and osmotic pressure (π) responses following step changes in water potential applied to the cambial surface of willow (Salix exigua Nutt.) bark strips. The kinetics of the turgor response were monitored with a pressure transducer. In separate experiments, the kinetics of the π response were followed by freezing point determinations on stylet exudate. The sieve tube volumetric elastic modulus in the bark strips was about 21 bars, but may be higher in intact stems. The membrane hydraulic conductivity was about 5 × 10⁻³ centimeters per second per bar; several factors make it difficult to estimate its value accurately. Differences in the turgor pressure (P) and π responses, as well as the relatively more rapid initial turgor response to a water potential (ψ) change, suggested a time-dependent component in sieve tube wall elasticity.

Our observations were generally not supportive of the idea that sieve tubes might osmoregulate. However, the bark strip system may not be suitable for addressing that question.

Separate measurements of ψ, P, and π demonstrate that the relationship predicted by the fundamental cell water potential equation, ψ = P − π, is applicable within experimental error (± 0.4 bar) to sieve tube water relations.

     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Investigation of Bidirectional Movement of Tracers in Sieve Tubes of Salix Viminalis L.

Sieve-tube sap was collected as honeydew from the aphid Tuberolachnussalignus (Gmelin) feeding on a young shoot or a mature stemof willow (Salix viminalis L.). Two radioactive tracers (either³H-glucose and ¹⁴C-sucrose, or ¹⁴CO₂, Na₂ ¹⁴CO₃, and ³²P-orthophosphate)were applied to leaves or to bark abrasions at each end of thestem. In some experiments the tracers were applied after thestart of honeydew production, whilst in others the tracers wereapplied before the aphids were sited. In most cases aphids feeding between the tracer applicationpoints produced doubly-labelled honeydew during a 24–48-hperiod from tracer application. In some instances the firstdrops of honeydew contained one tracer, followed several hourslater by the other tracer, whilst some aphids produced doubly-labelledhoneydew from the first drop. Singly-labelled or inactive honeydewwas found in only a few cases. It was demonstrated that when ³H-glucose and ¹⁴C-sucrose wereapplied at opposite ends of a stem that the tracers moved inopposite directions in the form of sucrose. The data suggest that a simultaneous bidirectional movementof two tracers may occur in the same sieve tube. However, thepossibility that the doubly-labelled honeydew could be producedby lateral movement from one sieve tube carrying one tracerto an adjacent punctured sieve tube carrying the other tracercannot be discounted.     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

HORMONAL REGULATION OF EPIPHYLLOUS BUD RELEASE AND DEVELOPMENT IN BRYOPHYLLUM CALYCINUM

The effects of various concentrations of several auxins and cytokinins separately and in combination on epiphyllous budding of Bryophyllum calycinum were investigated, using a marginal leaf strip method. The number of buds released per leaf in 10⁻⁶ m benzyladenine (BA) was 2.8 times greater than those in water, and 1.6 times greater in 10⁻⁵ m isopentyladenine (IPA). BA, IPA and kinetin were antagonistic to indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) when used in combination treatments. The IAA oxidase co-factor p-coumaric acid (PCA) increased the number of epiphyllous buds to twice that of the water controls. This stimulatory effect of PCA on bud release suggests that an increase in IAA oxidase activity may be one of the significant changes that precedes the release of epiphyllous buds.     

Translocation of Shoot-Applied Indolylacetic Acid into the Roots of Populus tremula

Application of 10 to 100 μg indol-3-ylacetic acid to the leaves of rooted cuttings of aspen caused inhibition of root growth after three hours. Root growth recovered within 24 hours after IAA treatment. Swelling of the root tips occurred during the period of inhibition. The roots responded in the same way if IAA was applied in solution to the cut stem surface above the mature leaves. IAA-1-¹⁴C applied through a cut stem surface or to mature leaves was translocated downwards in the plants and labelled IAA could be isolated from the roots 3 to 24 hours after application. The ethanol-soluble activity decreased rapidly indicating a rapid metabolism or binding of IAA. IAA-1-¹⁴C applied to growing leaves was not translocated. From the rapid response of root growth it was concluded that IAA was translocated into the roots at a rate of about 7 cm per hour. This rate of translocation indicates that the sieve tubes are involved in the translocation. Implications of the results for the translocation of endogenous auxin into the roots are discussed.     

Measurement of the sieve tube membrane potential

A procedure is described for the measurement of the sieve tube membrane potential in the phloem of bark strips from Salix exigua Nutt. Measurements were made by inserting a measuring microelectrode into sap exuding from severed stylets of the willow aphid, Tuberolachnus salignus. Data taken from 20 bark strips gave an average potential of −155 ± 9 millivolts. Evidence is presented for an electrogenic component of the sieve tube membrane potential. The occurrence of a saturable sucrose-induced membrane depolarization is consistent with the concept of sugar accumulation by a sucrose/H⁺ co-transport mechanism.     

Effects of phytohormones on some biochemical parameters during dark induced leaf senescence ofSechium edule on Darjeeling Hill of the Eastern Himalayas

All the concentrations (25-150 mgl-¹) of the phytohormones kinetin, IAA (indol-3-ylacetic acid) and GA₃ (gibberellic acid) increased the activity of DCPIP (2,6 dichlorophenolindophenol)-Hill reaction, chlorophyll and protein contents over the control data in leaves ofSechium edule Sw. on Darjeeling hill of the Eastern Himalayas; while ethrel (2-chloroethylphosphonic acid) treatments decreased these parameters in the hilly species. The most effective concentrations in increasing these parameters were 50 mg 1-¹ of kinetin, 50 mg 1-¹ of IAA and 100 mg 1-¹ of GA₃; whereas 50 mg 1-¹ of ethrel was most effective in decreasing these parameters during the induction of senescence in the hilly vegetable crop. The increase in these parameters was greatest with kinetin, followed by IAA and least with GA₃ in the hilly plant species studied.     

α-Amylase in the Barley Grain Influenced by the Auxin-Cytokinin Interaction in the Coleoptile

Two phases are distinguished in the α-amylase production in barley (Hordeum vulgare) grains. There is an increase in activity extended to the third or fourth day of germination, then a slight decrease follows. This decrease is accelerated by kinetin while it is prevented by IAA applied at the top of the embryo coleoptile. IAA reverses partially the kinetin action. IAA applied in the germination medium has practically no effect. Removal of the coleoptile stops further increase in α-amylase activity and induces complete insensitivity to hormone treatment. The results indicate that auxin metabolism in the coleoptile participates in the control of α-amylase evolution in the barley grain and that kinetin could act through auxin metabolism in this coleoptile.     

Transfer of exogenous auxin from the phloem to the polar auxin transport pathway in pea (Pisum sativum L.)

When [1-¹⁴C]indol-3yl-acetic acid ([1-¹⁴C]IAA) was applied to the upper surface of a mature foliage leaf of garden pea (Pisum sativum L. cv. Alderman), ¹⁴C effluxed basipetally but not acropetally from 30-mm-long internode segments excised 4 h after the application of [1-¹⁴C]IAA. This basipetal efflux was strongly inhibited by the inclusion of 3.10^–6 mol· dm³ N-1-naphthylphthalamic acid (NPA) in the efflux buffer. In contrast, when [¹⁴C] sucrose was applied to the leaf, the efflux of label from stem segments excised subsequently was neither polar nor sensitive to NPA. The [1-¹⁴C]IAA was initially exported from mature leaves in the phloem — transport was rapid and apolar; label was recovered from aphids feeding on the stem; and label was recovered in exudates collected from severed petioles in 20 mM ethylenediaminetetraacetic acid. No ¹⁴C was detected in aphids feeding on the stems of plants to which [1-¹⁴C]IAA had been applied apically, even though the internode on which they were feeding transported considerable quantities of label. Localised applications of NPA to the stem strongly inhibited the basipetal transport of apically applied [1-¹⁴C]IAA, but did not affect transport of [1-¹⁴C]IAA in the phloem. These results demonstrate for the first time that IAA exported from leaves in the phloem can be transferred into the extravascular polar auxin transport pathway but that reciprocal transfer probably does not occur. In intact plants, transfer of foliar-applied [1-¹⁴C]IAA from the phloem to the polar auxin transport pathway was confined to immature tissues at the shoot apex. In plants in which all tissues above the fed leaf were removed before labelling, a limited transfer of IAA occurred in more mature regions of the stem.Abbreviations IAA indol-3yl-acetic acid - EDTA ethylenediaminetetraacetic acid - NPA N-1-naphthylphthalamic acid We are grateful to the Nuffield Foundation for supporting this research under the NUF-URB95 scheme and for the provision of a bursary to A.J.C. We thank Professor Dennis A. Baker for constructive comments on a draft of this paper and Mrs. Rosemary Bell for her able technical assistance.     

Changes in the amino acid composition and protein modification in phloem sap of rice

Pure phloem sap was collected from insects feeding on rice (Oryza sativa L.) leaves by a laser technique similar to the aphid stylet technique. Rapid circulation of nitrogen in the sieve tubes was demonstrated directly using ¹⁵N as a tracer. Application to the roots of the metabolic inhibitors of amino acids, aminooxyacetate and methioninesulfoximine, changed the amino acid composition in the sieve tubes. Feeding methionine to leaf tips resulted in its bulk transfer into the sieve tubes. In vitro experiments confirmed the existence of protein kinases in the pure rice phloem sap. The phosphorylation status of the sieve tube sap proteins was affected by the light regime. The possibility that changes in chemical composition or protein modification such as phosphorylation in the sieve tubes might affect plant growth are discussed.Analysis of pure phloem sap collected from rice plants by insect laser technique has shown dynamic changes in the chemical composition and the quality of proteins in the sap.