Computer simulation of osmotic expansion and shrinkage in okra hypocotyl segments

Okra hypocotyl segments were incubated in solutions of 0.3 or 0.4 M sorbitol at various temperatures and their shrinkage was measured. The result yielded an apparent activation energy for shrinkage of 4.8 kcal/mol, which is close to that of the viscosity of water. This coincidence suggests that the viscosity of water, i.e., the reciprocal function of water conductivity, is a limiting factor for osmotic shrinkage. Abrasion of okra hypocotyl segments with Carborundum substantially increased the rate of their osmotic shrinkage, indicating that the cuticle is the major barrier to water uptake by segments. The apparent activation energy for osmotic shrinkage was 4.5 kcal/mol in abraded segments. By introducing water conductivity into an algorithm, osmotic shrinkage and expansion of hypocotyl segments was successfully predicted by computation with this algorithm. Hence the extent of the contribution of water conductivity in osmotic shrinkage and expansion can be evaluated. Based on this simulation, water conductivity was identified as one of the major factors in governing the elongation growth rate of cells along with the osmotic pressure of the cell sap and the mechanical properties of the cell wall.     

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.     

The Effect of Coumarin on Young's Modulus in Sunflower Stems and Maize Roots and on Water Permeability in Potato Parenchyma

By means of the resonance frequency method Young's, modulus has been determined after coumarin treatment of growing segments of etiolated sunflower hypocotyl segments and in maize roots. Coumarin caused a decrease in Young's modulus in both shoot and root tissue. The response was very rapid; in sunflower hypocotyls the decrease in elastic modulus appeared 3 min after application of coumarin. The effects produced by coumarin were similar to those found by auxin. Coumarin increased the rate of water efflux out of potato parenchyma by about 20%. The increase in water permeability was evident within 3 min.     

FORMATION OF AUXIN IN YEAST CULTURES

We have found far more auxin in the culture media of bakers'' yeast than was obtained by Kögl and Kostermans from the cells themselves. The production of auxin by yeast cells resembles the formation observed in other organisms such as Rhizopus and Rhizobium which also form auxins in their culture media. The auxin yield was found to increase with the concentration of sucrose and to decrease with the concentration of peptone. An inverse relation with the rate of cell multiplication was observed. Enlarged and elongated cells appeared only in those media which contained considerable amounts of auxin. The total auxin yield in the various cultures was found to be directly proportional, below pH 5, to the hydrogen ion concentration. Thus, it was proposed that certain growth conditions favor the breakage of the link between auxin and its protein carrier (Skoog and Thimann) 1940) and consequently accelerate the rate of excretion of auxin into the growth medium.     

水分浸泡过夜对刺槐枝条最大水分导度测定的影响及年龄差异

枝条最大水分导度是植物水分生理学研究中的核心指标之一，冲洗法是当前用来测定该指标最常用的方法，但在实验测定之前首先必须要找到获得稳定的最大水分导度的可靠方法。本研究中，我们以刺槐（Robinia pseudoacacia L.）不同年龄枝条为实验材料，通过枝条采集后立即用于实验测定和枝条经超纯水中浸泡过夜后再用于实验这两种不同处理方式对不同年龄枝条最大水分导度测定的影响，检测两种处理方式中枝条最大水分导度随冲洗时间的延长是否保持恒定来判断最大水分导度测定值的可靠性，探讨提高枝条最大水分导度测定结果稳定性的有效方法。结果显示：①未浸泡过夜的当年生枝条，经150 kPa压力冲洗达到最大导水率后，继续延长冲洗时间枝条导水率会呈近线性下降，而经超纯水浸泡过夜的当年生枝条以同样实验方法达到最大导水率后，继续延长冲洗时间枝条的导水率基本保持稳定。②二年生枝条无论是否经超纯水浸泡过夜处理，经150 kPa压力冲洗达到最大导水率后枝条的导水率都基本保持稳定，但经超纯水浸泡过夜后，二年生枝条达到最大导水率所需的冲洗时间明显比未浸泡处理短。③根据茎横截面上木质部面积所占百分比和枝条木质密度判断，实验期间当年生枝条的木质化程度明显比二年生枝条低。我们推测未经浸泡过夜的当年生枝条出现木质部水分导度随冲洗时间延长而下降是由于枝条遭受机械性损伤后产生的分泌物在冲洗过程中被引入导管堵塞导管腔所致，而超纯水浸泡枝条过夜可有效降低分泌物被引入导管的可能性。因此，我们认为超纯水浸泡枝条过夜，可提高枝条最大水分导度测定结果的稳定性。     

Phototropic bending of intact and wounded potato shoots

Intact and wounded shoots of in vitro cultured potato were investigated in situ to determine how their phototropic (PT) bending capacity was affected by water availability and exogenous auxin supplementation. Wounding strongly decreased PT bending but it recovered upon supplementation of water, auxin or both. Intact shoots required neither water nor auxin, while shoot segments required both. Shoot cuttings required only water, while, like shoot segments, decapitated shoots had a strong requirement for auxin. Water supplementation was beneficial in all treatments and PT bending was not affected in cultures that were submerged in water for a short period. Sucrose and inorganic salts present in the medium strongly affected PT bending capacity of cultures, favouring combinations with lower concentrations of both. Sucrose alone strongly promoted PT bending up to a concentration of 5%. Osmotic shock induced by the addition of small volumes of highly concentrated carbohydrate solutions (sucrose, glucose or sorbitol) induced a rapid but transient decline in PT bending capacity. These results indicate that water availability is a major factor that affects PT bending in potato plantlets.     

Effect of Peeling on IAA-induced Growth in Avena Coleoptiles

The act of peeling removes the epidermis exclusively from Avenacoleoptiles. Peeling inhibits IAA-induced growth, by inhibitingthe growth of segments incubated in the presence of IAA, andpromoting that of those incubated in water. The magnitude ofthe inhibition of IAA-induced growth is proportional to theamount of epidermis removed. It is shown that neither lateralswelling, wounding, anaerobiosis, nor exposure to supraoptimalconcentrations of IAA cause the inhibition. It is concludedthat in Avena coleoptiles the epidermis regulates the rate ofexpansion of the underlying parenchyma cells and is the principaltarget of IAA-action. Avena sativa L., oat, coleoptile, indol-3-ylacetic acid, auxin, extension growth     

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

Segments of mesocotyls of Avena sativa L. transported [1-¹⁴C]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.     

Onion root water transport sensitive to water channel and K+ channel inhibitors

Transroot osmotic water flux (Jos) and radial hydraulic conductivity (Lpr) in onion roots were greatly increased by three means; infiltration of roots by pressurization, repetition of osmosis and chilling at 5 degrees C. Jos was strongly reduced by the water channel inhibitor HgCl2 (91%) and the K+ channel inhibitor nonyltriethylammonium (C9, 75%), which actually made the membrane potential of root cells less sensitive to K+. C9 decreased the rate of turgor reduction induced by sorbitol solution to the same extent as HgCl2. Thus, C9 is assumed to decrease the hydraulic conductivity (Lp) of the plasma membrane by blocking water channels, although possible inhibition of the plasmodesmata of the root symplast by C9 cannot be excluded. Onion roots transported water from the tip to the base in the absence of the osmotic gradient. This non-osmotic water flux (Jnos) was equivalent to Jos induced by 0.029 M sorbitol. Jnos increased when Jos was increased by repetition of osmosis and decreased when Jos was decreased by either HgCl2 or by C9. The correlation between Jnos and Jos suggests that non-osmotic water transport occurs via the same pathways as those for osmotic water transport.     

Osmotic properties of pea internodes in relation to growth and auxin action

The water transport properties of etiolated pea (Pisum sativum L.) internodes were studied using both dynamic and steady-state methods to determine (a) whether water transport through the growing tissue limits the rate of cell enlargement, and (b) whether auxin stimulates growth in part by increasing the hydraulic conductance of the growing tissue.

Measurements using the pressure probe technique showed that the hydraulic conductivity of cortical cell membranes was the same for both slowly growing and auxin-induced rapidly growing cells (membrane hydraulic conductivity, about 1.5 × 10⁻⁵ centimeters per second per bar). In a second technique which measured the rate of water movement through the entire pea internode, the half-time for radial water flow was about 60 seconds and was not altered by auxin application. These results indicate that auxin does not alter the hydraulic conductance of pea stem tissue, either at the cellular or the whole tissue level.

Measurements of the turgor pressure of cortical cells, combined with osmotic pressure measurements of expressed cell sap, show that the water potential of growing pea stems was about −3 bars. When the growth rate was altered by various treatments, including decapitation, auxin application, cold temperature, and KCN treatment, the water potential was independent of the growth rate of the stem. We attribute the depression of the water potential in young pea stems to the presence of solutes in the cell wall free space of the tissue. This interpretation is supported by the results of infiltration and perfusion experiments.

From the results of these dynamic and steady-state experiments, we conclude that the internal gradient in water potential (from the xylem to the epidermis) needed to sustain cell enlargement is small (no greater than 0.5 bar). Thus, the hydraulic conductance of the tissue is sufficiently large that it does not control or limit the rate of cell enlargement.

     

Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

Effect of sorbitol addition on the physicochemical characteristics of starch–fatty acid systems

Aqueous maize starch dispersions (20%) were heated at 100 °C, in the presence of myristic, palmitic or stearic acid potassium salts as well as of sorbitol added at concentrations up to 60% (dry starch). Flow behaviour measurements at 100 °C indicated that interactions took place between the starch–fatty acid systems and sorbitol resulting in viscosity increase which was more pronounced as the sorbitol content increased. Water solubility measurements showed that a major part of sorbitol was easily extracted by excess water whereas sorption experiments revealed that the moisture uptake rate was proportional to sorbitol content of the starch systems examined. Thermomechanical studies indicated that the starch–fatty acid samples containing sorbitol up to 40% exhibited antiplasticizing behaviour. Scanning electron microscopy studies revealed that at sorbitol concentrations over 30%, free sorbitol crystals were formed on the surface of starch–fatty acid samples, whereas the percentage crystallinity as well as the crystallite size of samples were proportional to sorbitol content.     

Water permeability of human hematopoietic stem cells

It is currently impossible to isolate or identify human hematopoietic progenitor cells from the bone marrow, yet the biophysical properties of these cells are important for the development of techniques to isolate and preserve stem cells for transplantation. Osmotic permeability properties of human bone marrow stem cells were estimated from the kinetics of cell damage in a hypotonic solution measured using in vitro colony assays for multipotential (CFU-GEMM) and committed (BFU-E, CFU-GM) progenitor cells. Cells exposed to a hypotonic solution swell as a result of water influx, and the rate of change of volume is proportional to the hydraulic conductivity of the plasma membrane. Cell damage occurs when the cell volume exceeds the maximum tolerable volume, so the hydraulic conductivity can be estimated from the kinetics of cell damage. For all the progenitor cells studied, the mean value of the hydraulic conductivity was 0.283 micron3/micron2/min/atm at 20 degrees C, with an Arrhenius activation energy of 6.41 kcal/mole. No significant differences were observed in the osmotic properties of the various progenitor cells. These data were used to predict the osmotic responses of human bone marrow stem cells at subzero temperatures during freezing.     

Boron and calcium sites involved in indole-3-acetic Acid transport in sunflower hypocotyl segments

Sunflower (Helianthus annuus L. cv Russian Mammoth) hypocotyl segments deficient in either B or Ca exhibited a higher rate of potassium leakage, compared to nondeficient segments. Potassium leakage, used here as an indication of membrane integrity, was completely reversed by the addition of H₃BO₃ or Ca(NO₃)₂ to the incubation medium of the B-deficient or Ca-deficient hypocotyl segments, respectively. This role of B and Ca in membrane integrity, which may be important in the entry and exit of auxin in cells, is identified as the first site of action for each of these two essential elements in the basipetal secretion of auxin. A second site for B is postulated because auxin transport was not restored, even when K⁺ leakage has been completely reversed to the nondeficient level, when B-deficient hypocotyls were incubated in B solution. This lack of reversibility of auxin transport implied that the incubation for 2 h in B solution was not enough to restore the auxin transport process. However, since the transfer of B-deficient seedlings to B solutions prevented further deterioration of auxin transport, these observations suggest that: (a) either an intact seedling, or a longer period of incubation of the hypocotyl in B solution, is required for the synthesis or maintenance of the functional second site for B; (b) B is probably essential in the synthesis of a ligand, which may or may not be needed to bind B, but which is essential in the basipetal transport of auxin. The second site for Ca in auxin transport, is indicated by the complete reversal of its inhibition in Ca-deficient hypocotyl, when incubated in Ca solution. The second site for Ca is thought to be directly involved in the secretion of auxin, in which Ca probably plays the role of a second messenger, as in stimulus-response coupling. The two sites for Ca can be distinguished from each other by their cation specificity. The requirement for Ca in the first site can be substituted by other divalent cations, while the second site is highly specific for Ca.     

The Rate of Transport of Natural Auxin in Woody Shoots

A method is described for the estimation of the rate of movementand the quantity transported of the natural growth hormone instandard isolated segments of apple shoots. During controlledstorage diffusible auxin is collected, and later by dividingthe standard length of stem into small sections the locationof the auxin front is determined, from which the rate of transportis deduced. Temperature markedly affects both rate of transportand amount of auxin transported (cf. van der Weij, 1932), amaximum occurring at 27–30° C.; followed by a rapidfall to zero. The total diffusible auxin in a given length ofstem is not affected by storage temperatures below 30° C.but falls to zero at 42° C. The rate of transport and amounttransported are proportional to the oxygen tension over therange 0 to 5 per cent. O₂, and there is some evidence for destructionof auxin in tensions below 2 per cent.     

THE SWELLING OF ISOELECTRIC GELATIN IN WATER : II. KINETICS.

It has been assumed that gelatin consists of a network of an insoluble material enclosing a solution of a more soluble material. The swelling of gelatin is therefore primarily an osmotic phenomena in that the water tends to diffuse in owing to the osmotic pressure of the soluble material. This osmotic pressure is opposed by the elasticity of the insoluble constituent, and equilibrium results when these two pressures are equal. The rate of the entrance of water should then obey Poiseuille''s law, provided the variable terms are expressed as functions of the volume. Equations have been derived in this way which agree quite well with the experimental curves and which predict the proper relation between the size and shape of the block and the rate of swelling. They lead to a value for the rate of flow of water through gelatin which has been checked by direct measurement. The mechanism assumed predicts that at a higher temperature and under conditions such that the water has to pass through collodion before reaching the gelatin, the experiment should follow the same course as that of osmosis discussed previously. This was also found to be the case. The slow secondary increase in swelling is ascribed to fatigue of the elastic properties of the gelatin. The rate of this secondary swelling should therefore be independent of the size of the block, in contrast to the rate of primary swelling which is inversely proportional to the size. It can further be shown that this secondary swelling should be proportional to the square root of the time, and also that with large blocks at higher temperatures the entire swelling should be of this secondary type. These predictions have also been found to be true.     

Solutes in the free space of growing stem tissues

The concentration of osmotically active solutes in the cell wall free space of young stem tissues was studied using a variety of extraction methods. When the intercellular air spaces of etiolated pea (Pisum sativum L.) internodes were perfused with distilled H₂O, the resulting solution contained a solute concentration of about 70 milliosmoles per kilogram. A second procedure involving vacuum infiltration of segments followed by centrifugation to collect the free space solution gave similar results. Apical stem segments yielded free space extracts about twice as concentrated as those from basal portions of the stem. After correcting for dilution of the free space solution by the infiltrated water, the osmotic pressure of the undiluted free space in pea stem tissue was estimated to be 2.9 bars for apical segments, 1.8 bars for basal regions. These values may be somewhat overestimated due to solute efflux from intracellular pools during the extraction procedure. Similar results were obtained for stem regions of etiolated soybean (Glycine max [L.] Merr.) and cucumber (Cucumis sativus L.) seedlings.

From measurements of the electrical conductivity and refractive index of free space extracts before and after ashing, it appears that 25% of the solutes are inorganic electrolytes and 75% are organic nonelectrolytes with an average size similar to that of glucose.

A significant osmotic pressure in the wall space offers an explanation for the frequent observation that nontranspiring plants have negative water potentials. Calculations of hydraulic resistance from water potential data must take into account solutes in the free space, else `apparent,' but unreal, changes in resistance may be calculated.

     

INDUCTION OF WOUND-VESSEL DIFFERENTIATION IN ISOLATED COLEUS STEM SEGMENTS IN VITRO

Excised internodes and 2-mm-thick transverse stem segments of Coleus blumei were incubated 7 days on media containing 2% sucrose, 1% agar, and various growth substances. Wound-vessel members differentiated in the 2-mm-thick tissue slices incubated on medium containing no exogenous auxin (control). Compared to control slices, the addition to the medium of either IAA (50 or 5 ppm), 2, 4-D (10, 1, or 0.1 ppm), TIBA (50, 5, or 0.5 ppm), or kinetin (50, 5, 0.5 or 0.05 ppm) inhibited wound-vessel differentiation. Simultaneous treatment of tissue slices with IAA and kinetin inhibited wound-vessel differentiation, as did the incubation of tissue slices on medium containing no sucrose. Low concentrations of IAA (0.05 ppm) or 2, 4-D (0.01 ppm) resulted in over a 100% increase in the numbers of wound-vessel members differentiated. These results are interpreted as indicating auxin synthesis by the tissue slices and the participation of auxin as a limiting factor in xylogenesis. The inhibition of wound-vessel differentiation by relatively high concentrations of 2,4-D, TIBA, or kinetin is interpreted as a reflection of the inhibition of polar auxin transport by these substances, and an indication that polar auxin transport enhances xylogenesis.     

The hydraulic architecture of balsam fir (Abies balsamea)

Leaf-specific conductivities (LSCs – hydraulic conductivity per dry weight of supplied leaves). Huber values (transverse sapwood area per dry weight of supplied leaves), specific conductivity (hydraulic conductivity per transverse sapwood area) and tracheid diameters were measured throughout the trunk and crown of 20-year-old trees of Abies balsamca (L.) Mill. Measured specific conductivity was proportional to the radius to the fourth power of tracheids. LSCs, which indicate the relative water availability to different plant parts, are much higher in the trunk than in first order branches, and lowest in second order branches. The structural basis for this "hydraulic hierarchy" lies both in Huber values and in tracheid diameters. For similar diameter stem segments, there was no statistically significant difference for trunks versus branches in specific conductivity. However, in old parts of the tree, trunks are wider than supported branches and producer wider tracheids resulting in greater specific conductivities than in branches. In vigorous trees with strong apical control, Huber values were 12.0 times greater in the trunk than in similar diameter branch segments. In slow-growing trees with weak apical control, Huber values were 2.2 times greater in the trunk versus similar branch segments.     

Influence of Auxin on Young's Modulus in Stems and Roots of Pisum and the Theory of Changing the Modulus in Tissues

The effect of auxin on elastic extensibility has been investigated by means of the resonance frequency melhod in Pisum, sativum. The time lag for the decrease in Young's modulus E, caused by IAA, was between 2 and 3 minutes in etiolated stem internodes. The time lag for growth was about 7 minutes. The measurements of E in root segments were only qualitative owing to the structural characteristics; IAA decreases E in roots as it does in stems, but only in the region where IAA is assumed to enhance elongation. The connexion between elastic modulus and growth is discussed with reference to other investigations. The assumption has been made that a decrease in elastic modulus indicates a change in the cell wall which in some way is conducive to growth (induction of elongation). The theoretical possibilities of changing E have been discussed with reference to the formula for water fluxes. Both a change in a cell wall properly and a change in the cytoplasmic permeability are able to cause a change in E in the same way as auxin does. An early action of auxin must be located in the cell-wall-plasmalemma region.