Regulation of cell division and cell enlargement by turgor pressure

Isolated radish (Raphanus sativus L., var. Red Prince) cotyledons were incubated in growth medium plus graded concentrations of mannitol (−1 to −16 bars) for 28 hours. At the end of the incubation period, turgor pressures were measured using thermocouple psychrometers. Cell division, as measured by DNA increase, was greatly stimulated by increasing turgor from 5 to 6 bars. Cell enlargement was stimulated as turgor increased above 3 bars. The critical turgor pressure for increased cell division thus appeared significantly greater than that for increased cell enlargement.     

Promotion of radish cotyledon enlargement and reducing sugar content by zeatin and red light

Effects of zeatin on amino acid and sugar contents of detached radish (Raphanus sativus L.) cotyledons were investigated to determine if accumulation of these solutes contributes to cytokinin-enhanced growth. Protein and amino acid levels were not significantly affected, but in cotyledons incubated in light the hormone caused greater accumulations of reducing sugars than occurred in light controls. Continuous fluorescent light or a few minutes of red light increased both the growth rate and the reducing sugar levels compared to dark controls. A far red treatment following red light overcame the promoting effect of the latter. Amounts of reducing sugars were closely associated with growth under the above conditions. Activity of an unidentified amylase was elevated by continuous light or a red light treatment (nullifiable by far red), suggesting that reducing sugars were derived from starch. Zeatin-treated cotyledons exhibited less amylase activity than did light controls, perhaps implicating cytokinin-stimulated conversion of fats to sugars in light. In darkness zeatin promoted cotyledon growth but did not increase sugar levels nor amylase activity, suggesting that enhanced ion accumulation also contributes to the more rapid water uptake leading to growth.     

Estimation of osmotic parameters accompanying zeatin-induced growth of detached cucumber cotyledons

Water potential (ψ), the osmotic potential (ψ_π), and the pressure potential (ψ_p) of detached cotyledons isolated from Cucumis sativus L. cv Marketer seedlings after 0, 1.5, and 3 days growth with and without zeatin were determined. From zero time to 3 days, cotyledons incubated without exogenous zeatin exhibited a slight decrease in ψ (from −0.4 to −1.0 bars), while those grown with zeatin developed even more negative values (about −4 bars). Both groups showed rising ψ_π values (decreases in solutes per unit volume), but this rise was more dramatic in those treated with zeatin. These data indicate that the capacity of zeatin-treated cotyledons to take up water more rapidly than controls and thus expand faster must be due to wall loosening, as reflected in ψ_p values which declined during 3 days from about +11 bars to about +1.4 bars.

It was also found that freshly detached cotyledons or those grown without exogenous zeatin exhibited osmoregulation in polyethylene glycol (PEG) solutions. That is, while cotyledons initially lost H₂O into certain PEG solutions, their ψ values decreased over time and they began absorbing water after 1 to 4 hours. After 3 days growth, zeatin-treated cotyledons had lost most of this capacity of osmoregulate. It seems likely that osmoregulation in cotyledons not treated with zeatin is due to wall loosening rather than changes in ψ_π. Zeatin-treated cotyledons with already loosened walls may not have this option to deal with water stress and thus simply come to equilibrium with external PEG solutions.

     

Evaluation of h secretion relative to zeatin-induced growth of detached cucumber cotyledons

Cytokinins promote expansion of cotyledons detached from seedlings of more than a dozen species. The zeatin-enhanced expansion of cucumber (Cucumis sativus L. cv Marketer) cotyledons was investigated. In addition, whether acid secretion is involved in wall loosening accompanying such accelerated growth was evaluated. For cotyledons abraded with carborundum or cut into either eight or 18 pieces, we detected no zeatin-enhanced acidification of the growth medium during growth periods of 3 days. Measurements of pH values on each surface of zeatin-treated, abraded cotyledons after 3 days of growth also showed no detectable acidification caused by the hormone. Furthermore, with several buffers at pH values ranging from 5 to 8, growth of nonabraded, abraded, or cut cotyledons with or without zeatin was independent of external pH. However, experiments restricted to about 12 hours indicated that certain acidic buffers enhanced growth of cotyledons cut into 18 pieces. Lastly, concentrations of fusicoccin that caused growth promotion equal to that of zeatin initiated substantial acidification of the medium. Collectively, these data suggest that zeatin-induced expansion of detached cucumber cotyledons is independent of H⁺ secretion.     

Inhibition of Zeatin-Induced Growth of Cucumber Cotyledons by Sulfite Ions

The inhibitory effects of sulfite ions on zeatin-induced cellexpansion in cotyledons excised from dark-grown seedlings ofcucumber (Cucumis sativus L.) were examined. With 50 µMzeatin the growth rate in white light was about twice that ofthe control. Addition of Na₂SO₃ in the growth medium inhibitedthe zeatin-induced growth of cotyledons. Zeatin-treatment increasedthe osmotic potential in cell sap of cotyledons, while sulfitedecreased it. These treatments had no significant effect onpotassium concentration. Sulfite inhibited the zeatin-inducedincrease in contents of fructose and glucose, but did not affectsucrose content. The relative contents of non-cellulosic constituentsof cell walls fell with the advance of culture. This decreasewas repressed by sulfite, indicating that inhibition of expansiongrowth in cucumber cotyledons by sulfite ions was the resultof alterations in the cell wall structure due to changes inthe cell wall metabolism. (Received June 12, 1984; Accepted October 24, 1984)     

Salinity Stress Inhibits Bean Leaf Expansion by Reducing Turgor, Not Wall Extensibility

Treatment of bean (Phaseolus vulgaris L.) seedlings with low levels of salinity (50 or 100 millimolar NaCl) decreased the rate of light-induced leaf cell expansion in the primary leaves over a 3 day period. This decrease could be due to a reduction in one or both of the primary cellular growth parameters: wall extensibility and cell turgor. Wall extensibility was assessed by the Instron technique. Salinity did not decrease extensibility and caused small increases relative to the controls after 72 hours. On the other hand, 50 millimolar NaCl caused a significant reduction in leaf bulk turgor at 24 hours; adaptive decreases in leaf osmotic potential (osmotic adjustment) were more than compensated by parallel decreases in the xylem tension potential and the leaf apoplastic solute potential, resulting in a decreased leaf water potential. It is concluded that in bean seedlings, mild salinity initially affects leaf growth rate by a decrease in turgor rather than by a reduction in wall extensibility. Moreover, longterm salinization (10 days) resulted in an apparent mechanical adjustment, i.e. an increase in wall extensibility, which may help counteract reductions in turgor and maintain leaf growth rates.     

Response to osmotic medium and fusicoccin by seeds of radish (Raphanus sativus) in the early phase of germination

The effect of increasing osmotic values of the medium (mannitol) on the growth and the response mechanisms of seeds of radish ( Raphanus sativus L., cv. Ton do Rosso Quarantino) during the early phase of germination was investigated in the presence or absence of fusicoccin (FC). Decreasing the water potential in the medium inhibited the growth and the evolution of protein synthesis and enhanced H⁺ extrusion, net uptake of K⁺ and malic acid synthesis. FC, which stimulates these latter functions, counteracted the inhibitory effect of the decreasing water potential of the medium on growth and protein synthesis. Neither in the absence nor in the presence of FC did decreasing water potential of the medium enhance the synthesis of soluble sugars and amino acids to support the osmotic pressure of the seeds. The osmotic and water potentials of the seeds increased during germination. FC made the increase more rapid, while mannitol kept both potentials low. The pressure potentials of the seeds also decreased with time, and both FC and mannitol enhanced this change. If the seeds were without turgor, the development of protein synthesis was blocked. The seeds counteract the effect of decreasing water potentials in the medium by: a) enhancing H⁺ extrusion (and, as a consequence, wall loosening and transport mechanisms) and the synthesis of malic acid as apparent in the presence of FC; b) regulating the osmotic potentials of the cells (with a lower dilution of the osmotic compounds present in the seeds due to the diminished uptake of water); c) controlling the growth through the effects of a) and b) on the pressure potentials (internal hydrostatic pressure) of the seeds and on protein synthesis.     

Development and intracellular distribution of enzymes of the oxidative pentose phosphate cycle in radish cotyledons

Developmental and compartmentation studies were used to evaluate the relative roles of the oxidative pentose phosphate cycle, the Calvin cycle, and the glycolysis in cotyledons of radish (Raphanus sativus L.).     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Nitrate Reductase and Nitrite Reductase Activity in Dark-Grown Radish Seedlings: Relation to the Supply of NADPH

Functioning of nitrate reductase and nitrite reductase was measured in intact cotyledons from radish seedlings (Raphanus sativus L.) grown in the dark in a nitrate medium. Reduction of nitrate to nitrate did proceed during the whole period of 45 h, whereas the reduction of nitrite in the intact cotyledons dropped abruptly between 20 and 23 h after exposing the roots to nitrate. The activity of the enzymes glucose-6-P dehydrogenase (G6PDH) and 6-P-gluconate dehydrogenase (6PGDH), measured in cotyledon extracts, showed a sharp decline simultaneously with the drop in nitrite reductase activity of the intact cotyledons. It was concluded that the amount of NADPH generated by the enzymes G6PDH and 6PGDH is not sufficient to allow continuous functioning of nitrite reductase after 20 h in cotyledons of seedlings grown in the dark. Therefore, the results from our experiments point to the functioning of nitrite reductase as the rate limiting step in the reduction pathway of nitrate in the dark.     

Further Comparisons between Carbowax 6000 and Mannitol as Suppressants of Cucumber Hypocotyl Elongation

Stirring largely eliminated the greater effectiveness of Carbowax 6000 compared to mannitol in reducing cucumber (Cucumis sativus L.) hypocotyl section elongation. Stirring increased elongation in water but reduced elongation in growth regulator solution. Shrinkage of cells without plasmolysis in hypertonic Carbowax solutions indicates that Carbowax 6000 did not penetrate cell walls. Sections prevented from elongating during pretreatment exposure to saturated air grew as much as those not pretreated. Sections pretreated with isotonic Carbowax responded similarly, but sections pretreated with isotonic mannitol grew less.     

Morphactin effects on soybean leaf anatomy and chlorophyll content

The effect of chlorflurenol (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate) (CF) on chlorophyll (chl) content was studied in intact plants and floating leaf disks. For intact soybean (Glycine max (L.) Merrill) plants grown in the growth chamber, 2.5 μg/ml CF applied 10 to 20 d after planting retarded chl decline in senescing tissues such as cotyledons and unifoliate leaves and increased chl content in recently expanded tissues such as trifoliate leaves. CF did not retard chl decline in the dark unless regulator application was followed by a period of 24 h in the light prior to darkness. In floating leaf disk tests, CF retarded chl decline in dock (Rumex obtusifolius L.) and radish (Raphanus sativus L.) at concentrations of 10^?4 M, but was ineffective at lower concentrations. Chl decline was significantly hastened by CF in tobacco (Nicotiana tabacum L.) and soybean, but was unchanged in barley (Hordeum vulgare L.). CF treatment increased tissue weight (g fresh wt/cotyledon; g dry wt/ cm² for unifoliate and trifoliate leaves), decreased moisture content, and increased leaf thickness, palisade layer thickness, and palisade and spongy mesophyll cell counts. We conclude that plants treated with morphactins show greater green coloration predominantly because of growth effects, and only in small part because of prevention of chl decline in senescing tissues.     

Chromosaponin I increases mechanical extensibility of lettuce root cell walls

Chromosaponin I (CSI), at 3 m M , stimulates the growth of lettuce roots ( Lactuca sativa L. cv. Grand Rapids) with increasing fresh weight and decreasing root diameter compared with control. To analyze the mechanism of action of CSI, mechanical properties of lettuce root cell walls were examined with a tensiometer and the osmotic potential of the cell sap was measured with a vapor pressure osmometer. The mechanical extensibility of the cell wall was increased by CSI treatment, while the osmotic potential remained constant. Under osmotic stress, through addition of 0.225 M mannitol, the mechanical extensibility of the cell wall was increased before stimulation of growth was observed. These results suggest that cell wall-loosening is involved in the growth stimulation induced by CSI.     

Comparative effects of gibberellic acid and N6-benzyladenine on dry matter partitioning and osmotic and water potentials in seedling organs of dwarf watermelon

Apical applications of 0.2 μg N⁶-benzyladenine (BA), a synthetic cytokinin, or 5 μg of gibberellic acid (GA₃) significantly enhanced hypocotyl elongation in intact dwarf watermelon seedlings over a 48-h period. Accompanying the increase in hypocotyl length was marked expansion of cotyledons in BA-treated seedlings and inhibition of root growth by both compounds. A study on dry matter partitioning indicated that both growth regulators caused a preferential accumulation of dry matter in hypocotyls at the expense of the roots; however, GA₃ elicited a more rapid and greater change than did BA. In comparison to untreated seedlings, BA decreased total translocation of metabolites out of the cotyledons. Water potentials of cotyledons and hypocotyls were determined by allowing organs to equilibrate for 2 h in serial concentrations of polyethylene glycol 4000. Osmotic potentials were determined by thermocouple psychrometry. During periods of rapid growth in cotyledons and hypocotyls of BA-treated seedlings and in hypocotyls of GA-treated seedlings, the osmotic potential increased and the turgor pressure decreased in relation to untreated seedlings, indicating that cell wall extensibility was being increased. Osmotic potentials were lower in hypocotyls of GA-treated than in those of BA-treated seedlings, even though growth rates were higher in GA-treated seedlings, indicating that the latter treatment was generating more osmotically active solutes in hypocotyls.     

Effects of Compounds that Influence Calcium Absorption and of Calcium-Calmodulin Inhibitors on Zeatin Induced Growth and Chlorophyll Synthesis in Excised Cucumber Cotyledons

Previous results indicate that cytokinin action requires formationof Ca-calmodulin. We evaluated that hypothesis for cytokinin(zeatin) induced growth and chlorophyll formation in excisedcucumber (Cucumis sativus L.) cotyledons using several compoundsexpected to increase or decrease cytosolic levels of Ca²⁺ andthereby perturb formation of Ca-calmodulin. We also studiedeffects of eight drugs known to inhibit action of Ca-calmodulinin animals. Most of the results indicate no special requirementof Ca-calmodulin for cytokinin action in these organs. (Received November 24, 1988; Accepted April 24, 1989)     

Primary events regulating stem growth at low water potentials

Cell enlargement is inhibited by inadequate water. As a first step toward understanding the mechanism, all the physical parameters affecting enlargement were monitored to identify those that changed first, particularly in coincidence with the inhibition. The osmotic potential, turgor, yield threshold turgor, growth-induced water potential, wall extensibility, and conductance to water were measured in the elongating region, and the water potential was measured in the xylem of stems of dark-grown soybean (Glycine max [L.] Merr.) seedlings. A stepdown in water potential was achieved around the roots by transplanting the seedlings to vermiculite of low water content, and each of the parameters was measured simultaneously in the same plants while intact or within a few minutes of being intact using a newly developed guillotine psychrometer. The gradient of decreasing water potential from the xylem to the enlarging cells (growth-induced water potential) was the first of the parameters to decrease to a growth-limiting level. The kinetics were the same as for the inhibition of growth. The decreased gradient was caused mostly by a decreased water potential of the xylem. This was followed after 5 to 10 hours by a similar decrease in cell wall extensibility and tissue conductance for water. Later, the growth-induced water potential recovered as a result of osmotic adjustment and a rise in the water potential of the xylem. Still later, moderate growth resumed at a rate apparently determined by the low wall extensibility and tissue conductance for water. The turgor did not change significantly during the experiment. These results indicate that the primary event during the growth inhibition was the change in the growth-induced water potential. Because the growth limitation subsequently shifted to the low wall extensibility and tissue conductance for water, the initial change in potential may have set in motion subsequent metabolic changes that altered the characteristics of the wall and cell membranes.     

Hardening of root cell walls: a growth inhibitory response to salinity stress

Primary roots of intact maize plants (Zea mays L.) grown for several days in nutrient solutions containing 100 mol m⁻³ NaCl and additional calcium, had relatively inhibited rates of elongation. Possible physical restraints underlying this salt induced inhibition were investigated. The inhibition did not involve reductions in osmotic potential gradients and turgor in the tip tissues responsible for root elongation growth. The apparent yield threshold pressure, which is related to capacity of cell walls to undergo loosening by stress relaxation, was estimated psychrometrically in excised root tips. Salinity increased yield threshold values. Comparative root extensibility values were obtained for intact plants by determining the initial (1 min) increase in root elongation rate induced by an 0.1 MPa osmotic jump. Comparative extensibility was significantly reduced in the salinized root tips. Salinity did not reduce capacities for water efflux and associated elastic contraction in root tip tissues of intact plants exposed to hypertonic mannitol. We conclude that cell wall hardening in the elongating root tips is an important component of root growth inhibition induced by long-term salinization.     

An improved bioassay for cytokinins using cucumber cotyledons

The cucumber cotyledon greening bioassay is frequently used for detecting cytokinins. Beneficial modifications of the original technique included using 5-day-old cucumber (Cucumus sativus L., cv. National Pickling) cotyledons treated with combinations of 40 millimolar KCl and various concentrations of cytokinins. A dark incubation period of 20 hours was followed by an exposure to light for 3.5 hours. Under these conditions, extremely low (0.0001 milligram per liter) concentrations of N⁶-benzyladenine, zeatin, kinetin, or zeatin riboside can be detected. Of the four cytokinins tested, kinetin appeared to be the least active. With these improvements, the assay is 10 times more sensitive than is the previously described cucumber cotyledon greening bioassay for cytokinins.     

Soybean Seed Water Relations during in Situ and in Vitro Growth and Maturation

Water, osmotic, and pressure potentials of soybean (Glycine max [L.] Merrill) embryos and related maternal tissues were measured during periods of seed growth and maturation to test the involvement of embryo water relations in seed maturation. Seeds were matured in situ or in an in vitro liquid culture medium in detached pods or as isolated seeds. Changes in water relations of embryo tissues were independent of maternal tissues. During seed maturation in situ, water and osmotic potentials in both embryo and maternal tissues declined sharply near the time of maximum dry weight. During in vitro seed culture with and without pods, water and osmotic potentials in axis and cotyledon tissues declined continuously during growth. Water and osmotic potentials of the seed coat, which was present only during in vitro seed culture with pods, changed little during the culture period. Positive turgor in the embryo was maintained beyond maximum dry weight and the loss of green color during in vitro culture but declined to zero at maturity in situ. The osmotic potential in embryo tissues declined from −1.1 megapascals at early pod fill to between −1.65 and −2.2 megapascals at maximum seed dry weight across all maturation environments. It is suggested that the decreasing osmotic potential in the growing soybean embryo reaches a threshold level that is associated with cessation of growth and onset of seed maturation.     

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.