Basipetal Polarity Research Articles

Shoot regeneration process and optimization of Agrobacterium-mediated transformation in Sinningia speciosa

The florist’s Gloxinia, Sinningia speciosa, which bears considerable flower trait variations, is an emerging model plants to study floral traits development. However, the investigation of the genetic information linking these floral traits is limited due to a lack of a reliable and efficient transformation system for functional studies. This study aims to optimize a stable genetic transformation system for S. speciosa. Detailed regeneration process and tissue culture parameters are also elucidated. The results show that the plant regeneration, initiated from a single perivascular parenchyma cell, can be induced from leaf and petiole explants in the presence of 1 mg/mL 6-benzylaminopurine (BA) and 0.1 mg/mL naphthalene-acetic acid (NAA) through embryogenesis. In the presence of 0.1 mg/mL NAA only, the adventitious roots form prior to the re-differentiation of shoot tissues in leaf explants. When the proximal end of the petiole is orientated upright with the distal end to the medium, it results in higher success of regeneration, suggesting that hormone supplies must follow endogenous basipetal auxin polarity. Using a glucuronidase (GUS) reporter gene construct, maximum transformation (3.13%) was obtained after a 3 day pre-culture and 5 day co-culture from cotyledons and leaves of 3-week-old seedlings inoculating Agrobacterium strain EHA105. The putative transgenic lines were validated by RT-PCR, Southern blotting and GUS activity. Our result demonstrates that young seedlings are the best material for transformation, probably because young leaves are only a few cell layers thick allowing inner perivascular cell (the origin of regeneration) to be more accessible for Agrobacterium infiltration.

Actomyosin and movement in the angiosperm pollen tube: an interpretation of some recent results

Recent confirmations of the presence of myosin in angiosperm pollen tubes indicate that an energy-transducing actomyosin system is involved in the motility system of the vegetative cells. Myosin has been localised by immunofluorescence on the surfaces of vegetative nuclei and generative cells. It has been shown to be associated with individual amyloplasts in grass pollen, and there are indications that it is present on other particulate bodies in the cytoplasm. The organelles in the leading part of the tube move along separate traffic lanes of acropetal and basipetal polarity, known from electron microscopy and phalloidin labelling to contain numbers of fibrils containing aggregates of actin microfilaments; in older segments the movement can be related to single, uniformly polarised, fibrils. Circulatory flow is maintained at the proximal end by the looping of the fibrils in the grain or at callose plugs. Such loops do not occur at the apex, where entering organelles undergo random movement before becoming associated with basipetal streams. Vegetative nuclei and generative cells interact with several fibrils, and it is suggested that they are held in the leading part of the protoplast in unstable equilibrium between acropetal and basipetal forces. Constantly changing form, especially of the vegetative nucleus, is one consequence of these varying stresses. Possible analogies with the intracellular motility system of the giant cells of the Characeae are noted, and it is suggested that lipid globuli and other nonorganellar bodies may be transported in the pollen tube by association with myosin-bearing membranes similar to those involved in endoplasm movement in the characean cells.

Effects of cations on hormone transport in primary roots of Zea mays.

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and 3H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.

Adventitious Bud Formation from Bulb-scale Explants of Lilium speciosum Thunb. in vitro Effects of aminoethoxyvinyl-glycine, 1-aminocyclopropane-1-carboxylic acid, and ethylene

We studied the effects of AVG, ACC, and ethylene on the process of adventitious bud formation in vitro from bulb-scale explants of Lilium speciosum Thunb., cv. Rubrum nr. 10. AVG inhibited plantlet regeneration, especially at non-basal sites. The effects of AVG were counteracted by ACC and TIBA. Ethylene, applied in the first 3 or 7 days of the culture period in a concentration of 1 or 10 ppm, caused an increase in bud number per explant and suppressed the predominantly basipetal polarity of the regeneration sites. Ethylene increased the sensitivity of the tissue to exogenous auxin. A model is proposed showing the influence of ethylene, its biosynthetic pathway, and the other modifying factors on the regulation of plantlet induction in bulb-scale explants by auxin.

Strongly Acidic Auxin Indole-3-Methanesulfonic Acid

A radiochemical synthesis is described for [(14)C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [(14)C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles.

Adventitious Bud Formation From Bulb-scale Expiants of Lilium speciosum Thunb. in vitro. Effects of Wounding, TIBA, and Temperature

Effects of tissue wounding, TIBA, and temperature on the process of adventitious bud formation in vitro from bulb-scale expiants of Lilium speciosum Thunb., cv. Rubrum nr. 10, were studied. Removal of the abaxial epidermis from the expiants led to an increase in the number of plantlets formed at the adaxial surface and to suppression of the normally basipetal polarity of the regeneration process. It is concluded that the actual wounding itself was responsible for these effects and that wounding must have given rise to a transmittable stimulus. When incorporated into the nutrient medium, TIBA had an effect similar to that of wounding: the number of plantlets per expiant increased and the polarity was suppressed. However, at concentrations higher than 2.10-6M, TIBA inhibited the regeneration process. Within the range of 15°–25°C, a rise of the temperature led to more plantlets per expiant and to (partial) suppression of basipetal polarity. The results are discussed with special attention to the similarity in the action of these three factors.

Effects of osmotic stress on polar auxin transport in Avena mesocotyl sections

Segments of mesocotyls of Avena sativa L. transported [1-(14)C]indol-3yl-acetic acid (IAA) with strictly basipetal polarity. Treatment of the segments with solutions of sorbitol caused a striking increase in basipetal auxin transport, which was greatest at concentrations around 0.5 M. Similar effects were observed with mannitol or quebrachitol as osmotica, but with glucose or sucrose the increases were smaller. Polar transport was still detectable in segments treated with 1.2 M sorbitol. The effects of osmotic stress on the polar transport of auxin were reversible, but treatment with sorbital solutions more concentrated than 0.5 M reduced the subsequent ability of mesocotyl segments to grow in response to IAA. The increased transport of auxin in the osmotically stressed segments could not be explained in terms of an increased uptake from donor blocks. The velocity of transport declined with higher concentrations of osmoticum. The reasons for the enhancement of auxin transport by osmotic stress are not known.

Polarity of Indoleacetic Acid in young Coleus Stems

Young internodes of Coleus blumei Benth. have long been known for their sizable amount of acropetal indoleacetic acid movement. However, plants of the same clone, under improved growing conditions, now show almost absolute basipetal polarity of (14)C-indoleacetic acid, as measured by liquid scintillation counting of (14)C in the receiver cylinders of agar. The ratio of basipetal to acropetal movement is now as much as 85:1, instead of the 3:1 ratio found years ago under conditions providing slower growth.

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.

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.

Polarity of Thiamine Movement through Tomato Petioles

Thiamine-(14)C moved through petiolar sections of Lycopersicon esculentum Mill. var. Michigan State Forcing with striking similarity in kinetics to auxins and gibberellic acid moving through similar sections of other green plants. Thiamine moved with strong basipetal polarity, at a velocity of 3 to 5 mm per hour, and emerged unchanged into the basal receiver agar block, judging by chromatography. This lends support to the hypothesis that polar movement is a property of several classes of plant hormones, rather than being restricted to the auxins (as previously believed).

THE POLARITY OF AUXIN TRANSPORT IN INVERTED CUTTINGS

SummaryThe original, basipetal polarity of auxin transport persisted in the stems of inverted cuttings of Tagetes, tomato and tobacco in spite of the reversal of the relative positions of the roots and shoots. No significant acropetal auxin transport could be detected even after four months growth. These results indicate that the polarity of newly formed cells in secondarily thickening internodes is determined by the existing polarity of auxin transport within the tissues.

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport, in stelar tissues of Phaseolus coccineus roots

Movement of both [(3)H]GA1 and [(14)C]GA3 through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [(3)H]GA1 in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [(14)C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [(3)H]GA1 injected into cotyledons moved into and acropetally along the seedling root.

Polar Auxin Transport in Leaves of Monocotyledons

ALMOST nothing is known about the establishment of cellular polarity underlying the polar auxin transport system of higher plants. Osborne1 has suggested that the apical ends of cells derived from an apical meristem by sequential divisions are younger than the basal ends: their polarity and the basipetal transport of auxin are due to this age difference. Sachs2 in his work on regenerating vascular strands has found that gradients of auxin may be responsible for establishing the cellular polarity and the subsequent transport of auxin in the direction of the initial gradient. Shoot tips and expanding dicot leaves contain relatively high levels of auxin. The basipetal polarity of auxin transport in petioles and stems is therefore associated with basipetal auxin gradients. In grass coleoptiles the greatest amounts of auxin are found at the tip, where basipetal auxin transport is also associated with basipetal auxin gradients.

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.

Polar Movement of Indole-3-acetic Acid-14C in Roots of Lens and Phaseolus

A critical review of the few papers on IAA-(14)C movement in roots revealed apparent contradictions, as well as flaws in experimental design that would be apt to cause artifacts. The movement of (14)C from IAA-(14)C was studied in sections of Lens and Phaseolus roots, using a system 20 or more times as sensitive as any previously used. To make sure that our results with roots could be compared validly with published work on petioles and stems, we used the same techniques as we had earlier used for shoot structures. The results with Lens were similar in many ways to those for shoots: net movement into receiver blocks was very strongly polar, followed a linear course for several hours, and showed a velocity of the same order of magnitude as in shoots (and, in fact, very close in absolute value to that found in Coleus stem cylinders). Also, as with shoots, all the radioactivity in receiver blocks ran to the R(F) of IAA. The time-course of loss of counts from donor blocks was similar to that found in shoots. The 2 most striking differences from shoots were 1) the very low percentage of added (14)C that was moved into the receivers (about one-tenth of the values for bean petioles), and 2) the fact that the polar movement was acropetal in roots, rather than basipetal as in shoots. Results with Phaseolus roots were similar to those for Lens, although an additional complication with Phaseolus roots was the indication of a transitory stage of weak basipetal polarity in the first few hours after excising the section. This stage was followed in a few hours by a stronger acropetal polarity.

Auxin Polarity in the Coleus Plant

1. Transport of auxin in stems and roots of Coleus blumei Benth. was studied, using a new auxin assay technique, the pea-root test. This test is sensitive to auxin concentrations as low as 10-11 M and involves a positive stimulation of root growth by auxin. 2. Polar gradients exist in the Coleus plant, which run from a strict basipetal polarity in the vegetative stem apex to the opposite polarity in the root tip. 3. Flowering stems do not have a strict basipetal polarity but are capable of transporting auxin acropetally as well. 4. Evidence is presented suggesting that a diffusible substance or substances in the flowering apex can cause the incomplete polarity characteristic of flowering stems. 5. Phototropism experiments are reported which demonstrate that intact flowering stems transport the phototropic stimulus acropetally and that vegetative stems lack this capacity.

AUXIN–TRANSPORT IN THE HYPOCOTYL OF PHASEOLUS VULGARIS L.

AUXIN–TRANSPORT IN THE HYPOCOTYL OF PHASEOLUS VULGARIS L.