Timing of the auxin response in etiolated pea stem sections

The short term growth response of etiolated pea stem segments (Pisum sativum L., var. Alaska) was investigated with the use of a high resolution growth-recording device. The immediate effect of treatment with indole-3-acetic acid is an inhibition of growth. This inhibition lasts about 10 minutes, and then the rate of elongation rises abruptly to a new steady rate about 4 times the rate of elongation before auxin treatment. This rapid steady rate of elongation, however, continues for only about 25 minutes before declining suddenly to a lower steady rate of growth about 2 times the rate of elongation before the addition of auxin. Pretreatment of the segments with cycloheximide or actinomycin strongly inhibits both phases of auxin-promoted elongation without altering the length of the latent period in response to the hormone.     

The kinetics of bidirectional growth of stem sections from etiolated pea seedlings in response to acid,auxin and fusicoccin

Growth in length and diameter of abraded stem sections from etiolated pea (Pisum sativum L.) seedlings was monitored continuously using a double laser optical level auxanometer system. Acidic solutions (pH 4.0–4.5) induced rapid elongation accompanied by lateral shrinkage (up to 8% of the initial diameter). The shrinkage phase lasted for 30–45 min. Pretreatment with permeant solutes (KCl, NaCl, sucrose or glucose) prevented lateral shrinkage, while pretreatment with the impermeant solute, polyethylene glycol, did not block lateral contraction in response to acid. A slight turgor step-up given during the shrinkage phase inhibited lateral shrinkage and increased the elongation rate. Visual observation confirmed that shrinkage occurred and that the same region of the stem that contracted in diameter also elongated. It is proposed that lateral shrinkage results from a decrease in turgor pressure during acid-stimulated elongation. Elongation induced by auxin and fusicoccin (FC) was also accompanied by a decrease in the diameter; this decrease could be prevented by pretreatment with KCl or glucose. Thus, the early phase of auxin and FC action is acid-like. However, the shrinkage is of shorter duration (14–20 min) and it is less drastic (ca. 2%). In addition, FC caused lateral expansion after a 20-min lag period in stems pretreated with KCl. The results are consistent with an acid-growth mechanism during the early phase (first 20–40 min) of the responses to both auxin and FC. It is suggested that enhanced osmoregulation subsequently inhibits further lateral shrinkage and helps to maintain steady-state growth. FC, unlike auxin, may alter the anisotropic character of the wall.Abbreviations FC fusicoccin - IAA indole-3-acetic acid - LOLA laser optical levar auxanometer - PEG polyethyleneglycol 600     

Effect of gibberellic Acid on elongation and longevity of coleus petioles

The effects of gibberellic acid on the longevity and elongation of variously aged, debladed petioles of Coleus blumei were studied, with particular reference to the hypotheses 1) that auxin increases longevity by increasing growth, and 2) that gibberellic acid acts by increasing the endogenous levels of auxin.

Gibberellic acid, substituted for the leaf blades, significantly decreased longevity of younger petioles, as measured by days or hours to abscission. Gibberellic acid also decreased the longevity resulting from 0.1% indoleacetic acid. This is the opposite of the effect expected if it is increasing auxin levels in the petiole.

In its effect on elongation of younger petioles, however, gibberellic acid did act in the direction expected if it were increasing effective levels of auxin in the petiole. The elongation rate from 0.1% gibberellic acid plus 0.1% indoleacetic acid in lanolin was as large or larger than that for 1.0% indoleacetic acid.

Petioles which were 10 or more weeks old (i.e., at positions 5+ below the apical bud were not affected by 0.1% gibberellic acid in either longevity or rate of elongation, with or without 0.1% indoleacetic acid. Since 1.0% indoleacetic acid increases both longevity and elongation rate of these petioles over 0.1% indoleacetic acid, gibberellic acid is clearly not acting on older petioles as if it were increasing effective auxin levels).

     

Acropetal transport of kinetin in pea stem sections

Pomocí stimulace r?stu postranních pupen? etiolovaných ?ízk? hrachu kinetinem bylo zji?těno, ?e biologický ú?inek kinetinu není omezen pouze na místo jeho aplikace, ale ?e se m??e projevit i v jiných ?ástech ?ízk?. Tento jev si autor vysvětluje transportem kinetinu.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Ethylene modification of an auxin pulse in cotton stem sections

The effect of ethylene on the basipetal movement of indole-3-acetic acid-1-¹⁴C through cotton stem sections (Gossypium hirsutum, L. var. Stoneville 213) was studied apart from processes involved in the uptake and exit of auxin by the section. Stem sections 60 mm in length were pretreated with ethylene or placed in room air (control) and pulse labeled for 20 min with IAA-1-¹⁴C. In both the ethylene treated and control sections, the IAA-1-¹⁴C taken up moved basipetally as a peak of radioactivity. Generally, the applied pulse moved down the stem sections at an average velocity of approximately 5.8 mm per hr. In some experiments, however, ethylene slightly reduced the velocity of auxin transport. Although the peak of radioactivity became broader and more dispersed during its migration through the section, it was still distinguishable after 7 hr of transport.     

Methionine metabolism and ethylene formation in etiolated pea stem sections

Stem sections of etiolated pea seedlings (Pisum sativum L. cv. Alaska) were incubated overnight on tracer amounts of l-[U-(14)C]methionine and, on the following morning, on 0.1 millimolar indoleacetic acid to induce ethylene formation. Following the overnight incubation, over 70% of the radioactivity in the soluble fraction was shown to be associated with S-methylmethionine (SMM). The specific radioactivity of the ethylene evolved closely paralleled that of carbon atoms 3 and 4 of methionine extracted from the tissue and was always higher than that determined for carbon atoms 3 and 4 of extracted SMM.Overnight incubation of pea stem sections on 1 millimolar methionine enhanced indoleacetic acid-induced ethylene formation by 5 to 10%. Under the same conditions, 1 millimolar homocysteine thiolactone increased ethylene synthesis by 20 to 25%, while SMM within a concentration range of 0.1 to 10 millimolar did not influence ethylene production. When unlabeled methionine or homocysteine thiolactone was applied to stem sections which had been incubated overnight in l-[U-(14)C]methionine, the specific radioactivity of the ethylene evolved was considerably lowered. Application of unlabeled SMM reduced the specific radioactivity of ethylene only slightly.     

Effect of abscisic acid and auxin on ribonuclease during ageing of bean endocarp tissue sections

The activity of RNase increases rapidly upon cutting sectionsof bean (Phascolus vulgaris L. var. Kentucky Wonder) endocarp,peaks within 4 to 8 hr and then declines. This rapid developmentof RNase activity is inhibited by cycloheximide. Auxin (naphthaleneaceticacid, NAA) accelerates the rate of decline of RNase. Abscisicacid (ABA) enhances the level of RNase between 4 and 24 hr,associated with a decline in RNA, and this effect of ABA isobscured in the presence of auxin. ¹ This work was supported by National Science Foundation Grant(GB-8316) to J. A. Sacher. ² On leave from Laboratorio di Radiobiochimica ed EcofisiologiaVegetale, C. N. R., Roma, (Italy), with a Fellowship supportedby North Atlantic Threaty Organization. ³ Present address: Instituto di Botanica, Universita di Ban,Bari, 70126, Italy. (Received April 1, 1971; )     

Relationship of lipid metabolism to the respiration and growth of pea stem sections

Biologically active lipids increase the growth of pea stem sections within 3 hours at the same time their respiration is increased and their growth rate is more than that of the intact plant. The greater final length of the intact internode is due to a longer growth period.

Both active and inactive lipids are rapidly taken up and enter all major metabolic fractions: among centrifugal fractions methyl oleate tends to label those that contain metabolically active membranes. It is concluded that lipids active in the bioassay are probably the effective molecules at the subcellular site of action.

No direct effect of lipids on isolated mitochondria could be shown. The respiration of stem tissue was not influenced by dinitrophenol and carbonyl cyano m-chlorophenyl hydrazone although dinitrophenol inhibited growth. Lipid-induced respiration was sensitive to these agents as well as to cyanide, indicating cytochrome oxidase is probably involved.

The promotion of growth and respiration by lipids is not linked to protein synthesis, since actinomycin D, puromycin and cycloheximide failed to inhibit the respiratory increase even though strongly limiting amino acid incorporation into protein. It is most likely that the effect of lipids on growth is due to their promotion of respiration.

     

Uptake and metabolism of vitamins e and k by pea stem sections

The uptake of α-tocopherol and vitamin K₁ by pea stem sections is described. Vitamin K₁ appears to be stable within the plant tissue and is found distributed in all particulate cell fractions following uptake. Only a small proportion of the tocopherol taken up is recoverable and the majority of the compound appears to undergo catabolism.

The oxidation of tocopherols by a cell-free system is described. This system requires oxygen and appears to involve enzyme activity but does not appear to be linked with the action of lipoxidase.

     

Transport of exogenous auxin in two-branched dwarf pea seedlings (Pisum sativum L.)

Dwarf pea plants bearing two cotyledonary shoots were obtained by removing the epicotyl shortly after germination, and the patterns of distribution of ¹⁴C in these plants was investigated following the application of [¹⁴C]IAA to the apex of one shoot. Basipetal transport to the root system occurred, but in none of the experiments was ¹⁴C ever detected in the unlabelled shoot even after transport periods of up to 48 h. This was true both of plants with two equal growing shoots and of plants in which one shoot had become correlatively inhibited by the other, and in the latter case applied whether the dominant or subordinate shoot was labelled. In contrast, when [¹⁴C]IAA was applied to a mature foliage leaf of one shoot transfer of ¹⁴C to the other shoot took place, although the amount transported was always low. Transport of ¹⁴C from the apex of a subordinate shoot on plants bearing one growing and one inhibited shoot was severely restricted compared with the transport from the dominant shoot apex, and in some individual plants no transport at all was detected. Removal of the dominant shoot apex rapidly restored the capacity of the subordinate shoot to transport apically-applied [¹⁴C]IAA, and at the same time led to rapid cambial development and secondary vascular differentiation in the previously inhibited shoot. Applications of 1% unlabelled IAA in lanolin to the decapitated dominant shoot maintained the inhibition of cambial development in the subordinate shoot and its reduced capacity for auxin transport. These results are discussed in relation to the polarity of auxin transport in intact plants and the mechanism of correlative inhibition.Abbreviations IAA Indol-3-yl-acetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4D 2,4-dichlorophenoxyacetic acid - IAAsp Indol-3-yl-acetyl aspartic acid     

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.     

Transport of auxin (indoleacetic acid) through lipid bilayer membranes

Summary Diffusion of auxin (indole-3-acetic acid) through planar lipid bilayer membranes was studied as a function of pH and auxin concentration. Membranes were made of egg or soybean lecithin or phosphatidyl serine inn-decane (25–35 mg/ml). Tracer and electrical techniques were used to estimate the permeabilities to nonionized (HA) and ionized (A^–) auxin. The auxin tracer flux is unstirred layer limited at low pH and membrane limited at high pH, i.e., when [A^–][HA]. The tracer flux is not affected by the transmembrane voltage and is much higher than the flux predicted from the membrane conductance. Thus, only nonionized auxin crosses the membrane at a significant rate. Auxin transport shows saturation kinetics, but this is due entirely to unstirred layer effects rather than to the existence of an auxin carrier in the membrane. A rapid interconversion of A^– and HA at the membrane surface allows A^– to facilitate the auxin flux through the unstirred layer. Thus, the total flux is higher than that expected for the simple diffusion of HA alone. The relation between flux (J _A), concentrations and permeabilities is: 1/J _A=1/P ^UL([A^–]+[HA])+1/P _HA ^M [HA]. By fitting this equation to our data we find thatP ^UL=6.9×10^–4 cm/sec andP _HA ^M =3.3×10^–3 cm/sec for egg lecithin-decane bilayers. Similar membrane permeabilities were observed with phosphatidyl serine or soybean lipids. Thus, auxin permeability is not affected by a net surface charge on the membrane. Our model describing diffusion and reaction in the unstirred layers can explain the anomolous relationship between pH and weak acid (or weak base) uptake observed in many plant cells.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

Mycoflora of bean,broad bean,lentil, lupine and pea seeds in Saudi Arabia

Using the seed-plate method on glucose-Czapek's agar at 28 °C (±2 °C), 24 genera and 60 species in addition to two varieties were collected from bean, broad bean, lentil, lupine and pea seeds. The most frequent genera were Aspergillus (16 species + two varieties), Penicillium (14 species), Rhizopus (1 species) and Yeasts, followed by Fusarium (3 species), Mucor (4 species) and Drechslera (3 species). From the preceding genera, Aspergillus niger, A.flavus, Penicillium citrinum, Rhizopus stolonifer, Fusarium moniliforme or F. oxysporum, Mucor hiemalis or M. racemosus and Drechslera spicifera were the most prevalent species.     

Regulation of cell wall synthesis by auxin and fusicoccin in different tissues of pea stem segments

Cell wall synthesis was studied by determining the incorporation of [¹⁴C]-glucose into epidermal and cortical cell walls of etiolated Pisum sativum L. cv. Alaska stem segments. Walls were fractionated into the matrix and cellulose components, and incorporation into these components assessed in terms of the total uptake of label into that tissue. When segments were allowed to elongate, the stimulation of total glucose uptake by indole-3-acetic acid (IAA) and fusicoccin (FC) was greater than their stimulation of incorporation. IAA and FC thus did not stimulate precursor incorporation in elongating segments. When elongation was inhibited by calcium, however, IAA and FC significantly promoted wall synthesis in the cortex and vasular tissue (which shows almost no growth or acidification response to auxin). In these tissues incorporation into matrix and cellulose was promoted approximately equally. In the epidermis (thought to be the tissue responsive to auxin in the control of growth), FC promoted a significant increase in wall synthesis, although less than that in the cortex, while there was some evidence of a similar promotion by IAA. Both IAA and FC had a greater effect on incorporation into the matrix component of the wall than into cellulose. The results that FC caused a substantial promotion of cell wall synthesis which was not due solely to elongation, and that the inner non-growth responsive cortical tissues can respond to IAA. Moreover, a comparison of the effects of IAA and FC on the different components of the wall suggests that the response in the epidermis differs from that in the other tissues.