Donor Blocks Research Articles

Auxin transport in roots

The reasons underlying the initial increase and subsequent decrease in the amount of radioactivity in the receiver block at the apical end of a Zea root segment supplied with a basal donor block containing labelled IAA have been investigated.The phenomenon was observed in segments supplied with IAA-1-(14)C, IAA-2-(14)C and IAA-5-(3)H. An acropetal polarity in the movement of radioactivity into the receiver blocks was observed using donor blocks containing IAA-5-(3)H at concentrations as low as 10(-10)M.The decrease in the amount of radioactivity in the receiver block begins after 6-8 h of transport at 25° C, and is unaffected by renewal of the donor block every 2 h, or the presence of 2% sucrose in the donor and receiver blocks.The net export of radioactivity into the receiver block at the apical end of the segment virtually ceases after 6-8 h of transport at 25° C, and is not prolonged by the presence of 2% sucrose in the donor and receiver blocks. At 10° C, net export of radioactivity continues for at least the first 50 h of transport, and the amount of radioactivity in a continuously applied receiver block continues to increase over this period.Receiver blocks removed from the apical end of segments after 8 h of transport and placed on planchettes show little or no decrease in the amount of radioactivity they contain as a function of time, in marked contrast to those left in contact with the segment.There is a marked, and metabolically dependent, resorption of radioactivity from the receiver block at the apical end of the segment after about 8 h of transport at 25° C; most of the resorbed radioactivity remains in the apical 2-4 mm of the segment.There is a loss of radioactive CO2 from segments supplied with a basal donor block containing 10(-6)M IAA-1-(14)C at 25° C, the emission beginning after 6-8 h of transport. Segments similarly supplied with 10(-6)M IAA-2-(14)C did not begin to lose radioactive CO2 until after about 10-12 h of transport.The ability of the segments to transport radioactivity in a polar manner declines with time after they are excised from the root, regardless of whether their cut ends are kept in the intervening period in contact with plain agar blocks, or ones containing unlabelled IAA at 10(-6)M. By the 6th h after excision at 25° C no transport of radioactivity through the segments and into the receiver blocks could be detected in either the aropetal or basipetal direction.The decrease in radioactivity in the receiver block after transport periods of 6-8 h at 25° C is therefore due to (1) a cessation of net export of radioactivity into the block, and (2) the onset of a metabolically-dependent, net resorption of radioactivity. At this time substantial amounts of radioactive CO2 begin to be evolved from segments supplied with IAA-1-(14)C, whereas with IAA-2-(14)C radioactive CO2 is not evolved for a further 4-6 h.

Auxin transport in roots

The net uptake and movement of radioactivity by 12-mm root segments of Zea mays have been studied as a function of time at 5, 15 and 25° C. Segments were supplied with an agar donor block containing 1 μM IAA-1-(14)C or IAA-2-(14)C continuously or for a limited period of time (pulse-labelling). In the latter case the original donor block was replaced either by a plain agar block or by one containing 1 μM unlabelled IAA. Receiver blocks were placed at the other end of the segments.The net uptake of radioactivity from the donor block at 15° C was greater at the basal end than at the apical end of the segment. At 5 and 15° C, the net uptake from a basal donor was virtually linear with time but at 25° C the rate of net accumulation decreased after about 10 h. Decarboxylation of IAA undoubtedly occurred at 15 and 25° C when the concentration in the tissue attained a high value.An acropetally polarised movement of radioactivity into the receiver blocks occurred regardless of whether the results were based on the actual amounts of radioactivity in the receiver block, or on the amounts in the receiver block expressed as a percentage of the net total radioactivity accumulated from the donor block. Only one radioactive substance was present in the receiver block and it ran to the same Rf as IAA in the isopropanol: ammonium: water solvent system.The amounts of radioactivity moving into that part of the root segment at least 6 mm distant from the end in contact with either an apical or a basal donor block were assessed. An acropetal polarity in the movement of radioactivity was observed on the basis of the actual amounts of radioactivity in these distal parts of the segments, but no such polarity was evident when the amounts of radioactivity were expressed as a percentage of the net total accumulated from the donor block. At least 3 radioactive substances were present in the tissue in addition to the substance running to the same Rf as IAA. The distribution of radioactivity in the segment cannot therefore be used to assess the distribution of IAA.Acropetal movement of radioactivity into an apical receiver block is not dependent upon the continued uptake of IAA at the basal end of the segment. No distinct pulses of radioactivity were detected moving through the root segments.Only a small part of the radioactivity in the root segment appears to be located in the polar transport system, while the bulk is not. The polarity found in the movement of the bulk radioactivity within the segment seems to be related to the polarity in IAA uptake from the donor blocks.

Transport and Accumulation of IAA-14C in Tumour-formingNicotianaHybrids

The polar transport of indol-3yl-acetic acid (IAA-2-14C) in stem expiants and decapitated shoots of tumour-prone Nicotiana hybrids (2n, 3n, and 4n) was compared with that in the normal, non-tumorous parent species N. glauca and N. langsdorffii. The total uptake of the auxin from donor blocks was greatest in the hybrids and N. glauca. The velocity of the basi petal movement of IAA-14C was the same in all species tested, i.e. 8 mm/h. The transport capacity for the hormone, however, was decreased in the three tumour-prone hybrids. Gas chromatography showed that between 70 and 90 per cent of the transported auxin was present in the form of IAA, between 10 and 30 per cent in the form of indol-3yl-aldehyde (IAld). The basipetal transport exceeded the acropetal transport in young (third) internodes of all plants studied, whereas in older stem segments (tenth internodes) the reverse was found. The polarity of auxin transport was less well expressed in the tumorous hybrids. Blocking the active transport by pre-treatment of stem cuttings with 2,4-dinitrophenol (2,4-DNP) caused a drastic reduction in the polar IAA-14C movement ; in all plants tested the auxin transport was reduced to the same low level. The accumulation of auxin at the base of cuttings was higher in N. glauca and the 2n hybrid than in N. langsdorffii, i.e. about seven times higher after 1-h and three times higher after 12-h transport experiments. The release of 14C from the cutting into an agar receiver block, however, was markedly reduced in the 2n hybrid, whereas in N. glauca the labelled substances moved more freely into the receiver blocks. Differences in the capacity for the accumulation and the release of IAA-14C in hybrid and N. glauca stem tissues were studied using decapitated greenhouse plants wounded by incision above the fourth internode. Accumulation of the auxin occurred only above the wound cut in hybrid plants. This observation is consistent with the view that tumour formation on hybrid stems occurs at sites of wounding. Our data suggest an elevated auxin level to be present during tumour initiation at these sites. These results on polar transport and accumulation of IAA-14C in tumorous Nicotiana plants together with our previous data on various endogenous auxins suggest that the induction of neoplastic growth in tobacco plants is correlated with increased auxin levels and an accumulation of the hormone at sites of wounding.

Abscisic Acid Action on Basipetal Auxin Transport

AbstractSeveral experiments have been performed to analyse the ABA effects on the basipetal transport of IAA‐2‐14C, using sections of epicotyls prepared from etiolated Lens seedlings. The sections were incubated in an ABA solution or ABA was applied in the donor blocks containing IAA. For each type of assay, the uptake (analyses of the donor blocks) and the movement of IAA‐C14 (analyses of the receiver blocks) were inhibited by ABA. The distribution of continuous decrease of the radioactivity, along the sections' axis, showed a 14C level from the apical towards the basal segments. ABA caused a decrease in the 14C concentration for the total sections, but a relative increase for the basal segment. When ABA was applied simultaneously with IAA in the donor blocks, the transport velocity of IAA, through the sections, was not changed significantly, while an ABA pretreatment caused a significant decrease.

Sulfur Dioxide and IAA Transport in Isolated Bean Petiole Sections

The uptake and movement of IAA-l-Cl4 in 5 mm sections from the middle of petioles of primary bean leaves was studied under five concentrations of SO2 (0.0, 0.1, 1.0, 10.0 and 100.0 ppm) by suspending the sections horizontally between donor agar blocks containing C14 labeled IAA and plain agar receiver blocks and measuring radioactivity in the blocks after 6 hr by scintillation counting. Movement was strictly polar in a basipetal direction. There was no signiScant effect of SO2, petiole size) or interaction between these two variables on IAA movement or IAA loss from donor blocks. It is therefore unlikely that SO2 damage to plants results from any effect on IAA movement.

Polar Movement of Gibberellic Acid through Young Coleus Petioles

For several decades the only type of growth regulator believed to move with strong polarity through plants was the endogenous auxin indole-3-acetic acid (9, 15, 25). After it had been demonstrated that both naphthalene-acetic acid (8, 16) and the weed killer 2,4-D also moved with strong polarity when tested under the same conditions as IAA (17, 18), investigations were started on other types of growth regulators. Several cytokinins have been tested for polar movement through excised sections. The first reports of polarity in the movement of benzyladenine (1, 20) could not be confirmed in more detailed investigations by Fox and Weis (7). If kinetin moved at all through excised sections, it usually moved without polarity (14, 24). Adenine, a weak cytokinin, also moved without polarity through Coleus petiole sections (24). The two authors who have studied gibberellin movement through excised sections concluded that gibberellin moved through pea stems, but without polarity. Kato (13) used primarily ultraviolet absorption to estimate the acid extracted from receiver blocks of agar.3 Presumably forced to do so because of the insensitivity of this method, he used extremely high concentrations of GA34 in the donor blocks (1.4 grams per liter, in contrast to the milligram per liter concentration of GA3 typically used in physiological experiments). Clor (3), purporting to confirm Kato, added tritium-labeled gibberellin of unstated purity, concentration, or even structure and then counted the label extractable from tissue slices. No evidence was presented that any of the label thus counted was still with the gibberellic acid. Basipetally polar movement has been reported for both synthetic and natural abscisic acid isolated from tomato fruits in transport experiments with explants, petiole, and internode segments of Coleus rhenaltianus (6). Milborrow (19) has stated that abscisic acid labeled with radioisotope moves with 3:1 basipetal polarity through petiolar sections. By analogy with the active transport systems studied in cell physiology (22), we could expect more than just two plant hormones to show polar movement. The two negative reports with GA3 did not convince us that this expectation was unfoundedClor's because of its cited inadequacies, and Kato's because the levels of GA3 in the donor were so high. (The polarity of IAA

Auxin Transport in Roots

The effects of temperature on the polar movement of IAA through 6-mm and 12-mm segments of Zea mays roots have been investigated over the range from 1 to 50°C. At all temperatures an acropetal polar movement of IAA predominated, although at low temperatures and at 50°C the 6-mm segments showed a transient basipetal polarity, before the persistent acropetal polarity developed. At 1°C the differences between acropetal and basipetal movement of IAA were less distinct than at the other temperatures. There is, however, a marked metabolically-dependent acropetal movement of IAA through the tissues at 1°C, because when the segments were deprived of oxygen the acropetal movement was severely reduced while the basipetal movement was reduced to a smaller extent. At 1°C and at 5°C there was always a persistent basipetal polarity of IAA movement through 6-mm and 12-mm segments under anaerobic conditions. The velocity of acropetal movement (mm h−1) was the same through the 6-mm and the 12-mm segments and was markedly affected by temperature. It increased from 1°C to a maximum value of 8 mm h−1 at 31°C and then decreased again at 40 and 50°C. The velocity of basipetal movement could be assessed only at 1 and 5°C at which temperatures it was greater than the velocity of acropetal movement, and virtually independent of segment length. The acropetal flux of IAA (cpm h−1) was much less through 12-mm segments than through 6-mm segments. For both lengths of segment, however, the flux showed a complex relationship with ambient temperature, increasing from 1°C to a maximum at 10–15°C, declining to a minimum value at 31°C and then rising again at 40 and 50°C. The basipetal flux of IAA could be astimated only at 1 and 5°C at which it was very much smaller than the acropetal flux. The amount of IAA in the receiver blocks increased linearly with time at the lower temperatures. At temperatures within the range 15°C to about 31°C, however, the amount of IAA in the receiver blocks began to decline if the transport periods exceeded a certain length. The time at which this decline in the IAA in the receiver block began was related to the ambient temperature. Chromatographic analysis indicated one radioactive substance in receiver blocks at the apical end of segments supplied with IAA-1-14C at the basal end after transport periods of 6 h at 25°C, and 72 h at 5°C. The Rf of this substance was closely similar to that of the radioactive IAA supplied in the donor blocks.

Movement and Metabolism of Kinetin-14C and of Adenine-14C in Coleus Petiole Segments of Increasing Age

To see if polar movement was typical of growth-regulators other than auxins, the movement of adenine-8-(14)C and of kinetin-8-(14)C was studied in segments cut from petioles of increasing age. No polarity was found. In time-course experiments lasting 24 hr, kinetin showed a progressive increase of radioactivity in receiver blocks, while adenine showed a maximum at 8 hr with a decline thereafter. More kinetin moved through older segments than through younger ones. There was no difference in net loss as far as the position of the donor block is concerned. However, the loss of radioactivity from adenine donor blocks was much higher than the loss of radioactivity from kinetin donor blocks.The radioactivity in receiver blocks after 24 hr treatment with kinetin-(14)C was still with kinetin, judging by location on chromatograms. By the same criterion, adenine and a smaller amount of some other compound were in receiver blocks after a 6 hr transport with adenine-(14)C in the donors. By contrast, more zones of radioactivity were extracted from petiole segments to which kinetin or adenine had been added. For both purine derivatives the original compound represented no more than 20% of the total radioactivity extracted from the tissue after a transport period of 24 hr.

Transport and Metabolism of Indole-3-Acetic Acid in Coleus Petiole Segments of Increasing Age

Transport and metabolism of IAA-1-(14)C in Coleus blumei Benth. was studied by means of a combination of liquid scintillation counting, autoradiography and thin-layer chromatography. Transport of IAA in petiole segments of increasing age (No. 2-8) was strictly polar in a basipetal direction. No acropetal movement occurred in either young or old tissues. The greatest amount, expressed as a percentage of the radioactivity lost from the donor block, was found in basal receivers on petiole number 2. There was gradually less transport in older segments. The recovery as a percentage of the radioactivity not accounted for by donor and receiver blocks, measured by counting the radioactivity in an acetonitrile-extract of petiole segments, was low: 25 to 50%. In this acetonitrile-soluble fraction evidence for different radioactive compounds was found, depending on the age of the tissue. A possible relationship between the amounts of auxin transported in the tissue and its corresponding metabolism is discussed.

THE MOVEMENT OF IAA‐C14 IN THE HYPOCOTYL OF PHASEOLUS VULGARIS

The amount of IAA‐C14 transported basipetally through excised hypocotyl sections was strongly affected by the pH of the donor blocks, less so by the pH of the receivers. The effect of donor pH was mostly on uptake. The small amount of acropetal movement was not noticeably affected by pH. Sucrose added to the donor resulted in increased basipetal transport. The time‐course of C14 movement into basal receivers followed a linear course from 1.5 to 3 hr as expected, but there was no net loss from the donors until after 30‐45 min. The usual type of velocity calculation, which assumes uptake starting from zero time, would therefore be lower than the true value. Basipetal transport through segments cut from various positions in the hypocotyl and from seedlings of various ages was maximal in 6‐8‐day‐old hypocotyl segments cut 25‐30 mm below the cotyledons. Acropetal movement was minimal at all positions of all ages tested.

The Movement of IAA-C 14 in the Hypocotyl of Phaseolus vulgaris

The amount of IAA-C14 transported basipetally through excised hypocotyl sections was strongly affected by the pH of the donor blocks, less so by the pH of the receivers. The effect of donor pH was mostly on uptake. The small amount of acropetal movement was not noticeably affected by pH. Sucrose added to the donor resulted in increased basipetal transport. The time-course of C14 movement into basal receivers followed a linear course from 1.5 to 3 hr as expected, but there was no net loss from the donors until after 30-45 min. The usual type of velocity calculation, which assumes uptake starting from zero time, would therefore be lower than the true value. Basipetal transport through segments cut from various positions in the hypocotyl and from seedlings of various ages was maximal in 6-8-day-old hypocotyl segments cut 25-30 mm below the cotyledons. Acropetal movement was minimal at all positions of all ages tested.

Auxin transport in roots

Light promotes the net acropetal movement of (14)C through 6-mm subapical segments of dark-grown roots of Zea mays supplied at their basal ends with 1 μM IAA-1-(14)C in agar blocks. This promotion occurs only when the segments are irradiated during the transport period, and both red and blue light appear to be as effective as white light at the radiant flux densities used in this investigation. The promotion is not found if the segments are pretreated with light and then returned to darkness before the trasport of IAA-1-(14)C is determined. The very slight basipetal movement of (14)C through the segments supplied with an apical source of IAA-1-(14)C is unaffected by light.Only one radioactive substance is found in the apical receiver blocks. This substance has an Rf virtually identical to those of the stock solution of IAA incorporated into the donor block and of unlabelled IAA. The movement of radioactivity into the receiver blocks through, the illuminated segments therefore appears to reflect the movement of IAA. Light thus increases the acropetal movement of IAA through the Zea root segment.The primary roots of Zea mays var. Giant Horse Tooth seedlings grown in total darkness do not exhibit a positive geotropic response. When the seed is orientated with the embryo uppermost the radicle grows out horizontally. On exposure to light, however, the roots bend down. This reaction appears about 3-9 hours after the onset of illumination, and white, red and blue light appear to be equally effective at the flux densities employed in this study. Green light in the spectral band between 510-530 nm did not appear to induce this positive geotropic responsiveness.

Auxin transport in roots

The movement of IAA has been investigated in roots of dark-grown seedlings of Zea mays using IAA-I-(14)C.With 6-mm segments excised 1 mm below the apex of the root it has been shown that: (a) There is a strictly acropetal flux of IAA through the tissues, the amount of IAA found in an apical receiving block increasing almost linearly with increasing transport period up to about 6-7 hours, but thereafter declining for at least a further 18 hours. The onset of this decline appears to be dependent upon the concentration of IAA in the donor block. (b) The amount of IAA recovered in the apical receiving block increases with increasing concentration of IAA in the donor block over the range from 0.1-10 μM, with transport periods of both 4 and 9 hours. (c) The radioactivity in the receiving block is confined to the IAA molecule. (d) The orientation of the segment with respect to gravity did not significantly affect the acropetal polar flux of IAA in the tissue.With non-decapitated 7-mm root apices it has been found that the presence of the apex has no effect on the strictly acropetal flux of IAA in the tissues, but that it entirely prevented the emergence of IAA into an apical receiving block.

On the relation between auxin transport and auxin metabolism in explants of Coleus

Transport and metabolism of naphthylacetic acid, labelled with (14)C or with (3)H, were studied by means of the liquid scintillation counting technique in combination with thin layer chromatography.The amounts of radioactivity reaching the receiver blocks as well as the loss from donor blocks greatly depended on the donor concentration. The relative amounts in receiver blocks increased with decreasing auxin concentrations in donor blocks. This phenomenon may be ascribed to the low immobilization capacity of the tissue at very low auxin concentrations.The relative amounts of radioactivity lost from donor blocks increased with decreasing auxin concentrations in donor blocks.Different characteristics of auxin transport can be explained by assuming a movement in symplast or in apoplast. During transport in the symplast the auxin is immobilized. Auxin immobilization governs many characteristics of auxin transport and could have a regulating effect on the free auxin content in plant tissues.

TRANSPORT, IMMOBILIZATION AND LOCALIZATION OF NAPHTHYLACETIC ACID-1-14C IN COLEUS EXPLANTS

Summary The transport and metabolism of naphthylacetic acid −1-14C in explants of Coleus was studied by means of the liquid scintillation counting technique in combination with thin layer chromatography. Microautoradiography was used to study the intracellular localization of the auxin. The gross radioactivity in the tissue is mainly fixed just below the donor block. In this part of the explant the radioactivity is present in the cytoplasm next to the cell wall. Although all the radioactivity in the receiver block represents the auxin itself, tissue extracts showed an intense metabolic turnover of the auxin. A study was made of the time-dependency of the production of several compounds and the ratio between the compounds in extracts of different parts of the explant. Finally, the chemical identity of the substances is discussed.

MOVEMENT OF GROWTH REGULATORS IN PLANTS

SummaryThe movement of radioactivity added in the form of indoleacetic acid (as the natural hormone) or as 2,4‐dichlorophenoxyacetic acid (as a synthetic growth regulator) was followed in segments cut from petioles of Phaseolus vulgaris.The movement of 14C added as IAA was strongly basipetally polar, although significant amounts moved acropetally during 24 hours. The velocity of basipetal movement was 6 mm/hour for IAA‐[14C], and was the same for donor concentrations of 5 or 50 μM. The flux increased with increasing concentration in the donor blocks, but a progressively smaller percentage of the applied radioactivity was transported.2,4‐D‐[14C] was found to have very similar transport properties to those of IAA‐[14C], except that its velocity of basipetal movement was much less, namely 1 mm/hour. After correction had been made for the much slower net loss of 2,4‐D from its donor blocks, the acropetal movement of the 14C from the two substances was essentially the same.

MOVEMENT OF GROWTH REGULATORS IN PLANTS

SummaryThe synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D), labelled with carbon‐14 in the carboxyl group, was applied in blocks of agar gel to either the apical or the basal ends of segments cut from the petioles of young primary leaves of Phaseolus vulgaris, and the carbon‐14 appearing in receiving blocks at the other end was measured. When application was made to the apical ends, all the carbon‐14 accumulating in the receiving blocks appeared to be still in the form of 2,4‐D. This basipetal transport had a velocity of 0.6–1.0 mm/hour, and continued even when the concentration of 2,4‐D in the receiving blocks had become higher than that remaining in the donor blocks. Acropetal movement was much smaller. The ratio of acropetal to basipetal movement was approximately constant with time at low concentrations of 2,4‐D. At a high concentration, the ratio was increased; with longer segments it was decreased. More 2,4‐D entered the segments from blocks applied to the apical than to the basal ends. It is concluded that 2,4‐D is moved through these petiole segments by active polar transport.