Water Relations of Growing Maize Coleoptiles : Comparison between Mannitol and Polyethylene Glycol 6000 as External Osmotica for Adjusting Turgor Pressure

Water relations of growing segments of maize (Zea mays L.) coleoptiles were investigated with osmotic methods using either mannitol (MAN) or polyethylene glycol 6000 (PEG) as external osmotica. Segments were incubated in MAN or PEG solutions at 0 to - 15 bar water potential (Ψ_o) and the effects were compared on elongation growth, osmotic shrinkage, cell sap osmolality (OC), and osmotic pressure (π_i). The nonpenetrating osmoticum PEG affects π_i in agreement with Boyle-Mariotte's law, i.e. the segments behave in principle as ideal osmometers. There is no osmotic adjustment in the Ψ_o range permitting growth (0 to −5 bar) nor in the Ψ_o range inducing osmotic shrinkage (−5 to −10 bar). Promoting growth by auxin (IAA) has no effect on the osmotic behavior of the tissue toward PEG. In contrast to PEG, MAN produces an apparent increase in π_i accompanied by anomalous effects on segment elongation and shrinkage leading to a lower value for Ψ_o which establishes a growth rate of zero and to an apparent recovery from osmotic shrinkage after 2 hours of incubation. These effects can be quantitatively attributed to uptake of MAN into the tissue. MAN is taken up into the apoplastic space and the symplast as revealed by a large temperature-dependent component of MAN uptake. It is concluded that MAN, in contrast to PEG, is unsuitable as an extemal osmoticum for the quantitative determination of water relations of growing maize coleoptiles.     

Solutes contributing to osmotic adjustment in cultured plant cells adapted to water stress

Osmotic adjustment was studied in cultured cells of tomato (Lycopersicon esculentum Mill cv VFNT-Cherry) adapted to different levels of external water potential ranging from −4 bar to −28 bar. The intracellular concentrations of reducing sugars, total free amino acids, proline, malate, citrate, quaternary ammonium compounds, K⁺, NO₃⁻, Na⁺, and Cl⁻ increased with decreasing external water potential. At any given level of adaptation, the maximum contribution to osmotic potential was from reducing sugars followed by potassium ions. The sucrose levels in the cells were 3- to 8-fold lower than reducing sugar levels and did not increase beyond those observed in cells adapted to −16 bar water potential. Concentrations of total free amino acids were 4- to 5-fold higher in adapted cells. Soluble protein levels declined in the adapted cell lines, but the total reduced nitrogen was not significantly different after adaptation. Uptake of nitrogen (as NH₄⁺ or NO₃⁻) from the media was similar for adapted and unadapted cells. Although the level of quaternary ammonium compounds was higher in the nonadapted cells than that of free proline, free proline increased as much as 500-fold compared to only a 2- to 3-fold increase observed for quaternary ammonium compounds. Although osmotic adjustment after adaptation was substantial (up to −36 bar), fresh weight (volume increase) was restricted by as much as 50% in the adapted cells. Altered metabolite partitioning was evidenced by an increase in the soluble sugars and soluble nitrogen in adapted cells which occurred at the expense of incorporation of sugar into cell walls and nitrogen into protein. Data indicate that the relative importance of a given solute to osmotic adjustment may change depending on the level of adaptation.     

Growth and water relations of cultured tomato cells after adjustment to low external water potentials

Cultured cells of tomato, Lycopersicon esculentum Mill. cv VFNT-cherry, have been selected for resistance to water stress (low water potential) imposed by the addition of polyethylene glycol to the culture medium. The ability of nonselected cells to grow in media with low water potentials changes dramatically with the age of the cells (with respect to days following inoculation) whereas there is little effect of the age of selected cells on growth over the same media water potentials. The increased resistance of selected cells has limited stability in the absence of stress, indicating that resistance is established by a slow reversible adaptive process.

Increased resistance (growth) in the presence of water stress appears to result from considerable osmotic adjustment by the cells. Growth cycle-dependent changes in resistance of nonselected cells are correlated with osmotic potential changes which are associated with the normal cell growth pattern in culture. Lowered osmotic potential is maintained by selected cells throughout the entire growth cycle and may explain the growth cycle independence of growth of selected cells on polyethylene glycol-containing media. Osmotic adjustment of resistant cells at stationary phase can be as much as 40 bar. Turgor is maintained by resistant cells (as high as 21 bar) in media with low water potentials at least partly at the expense of cell expansion.

     

Growth and Nodulation Responses of Rhizobium meliloti to Water Stress Induced by Permeating and Nonpermeating Solutes

Isolates of Rhizobium meliloti, representing antigenically distinct indigenous serogroups 31 and 17, were grown in yeast extract-mannitol broth (YEM) containing NaCl or polyethylene glycol (PEG) to provide external water potentials ranging from −0.15 to −1.5 MPa. Several differences were found between representatives of the two groups in their abilities to adapt to water stress induced by the nonpermeating solute PEG. At potentials below −0.5 MPa, strain 31 had a lower specific growth rate than strain 17 and an irregular cell morphology. In contrast, neither growth nor cell morphology of either strain was affected significantly over the same range of water potentials created by a permeating solute, NaCl. Despite the superior growth of strain 17 at the low water potentials imposed by PEG, upshock of water-stressed cells (−1.0 MPa; PEG) into normal YEM (−0.15 MPa) resulted in a faster recovery of growth by strain 31 than by strain 17. Different responses of the two strains to a water potential increase were also revealed in nodulation studies. Strain 31 required significantly fewer days to nodulate alfalfa than strain 17 did when the strains were transferred from YEM with PEG at −1.0 MPa onto the roots of alfalfa seedlings in plant growth medium (−0.1 MPa). The addition of supplemental calcium (0.1 mM) to growth medium with PEG (−1.0 MPa) reduced the differences between strains in their responses to water stress. The severe growth restriction and morphological abnormalities shown by strain 31 were corrected, and the prolonged recovery time shown by water-stressed cells (−1.0 MPa; PEG) of strain 17 upon transfer to normal YEM was shortened. The latter strain also nodulated earlier and more rapidly after growth in PEG medium at −1.0 MPa in the presence of supplemental calcium ions. These results indicate that the efficacy of osmoregulation can vary among strains of the same species and that the mechanism of osmoregulation may differ depending on the nature of the water stress.     

Differential Effects of Permeating and Nonpermeating Solutes on the Fatty Acid Composition of Pseudomonas putida

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that −1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas −1.0-MPa NaCl-treated or control cells did not. At the range of water potential (−0.25 to −1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the −0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than −0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in −1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.     

Stress-induced osmotic adjustment in growing regions of barley leaves

Young barley seedlings were stressed using nutrient solutions containing NaCl or polyethylene glycol and measurements were made of leaf growth, water potential, osmotic potential and turgor values of both growing (basal) and nongrowing (blade) tissues. Rapid growth responses similar to those noted for corn (Plant Physiology 48: 631-636) were obtained using either NaCl or polyethylene glycol treatments by which exposure of seedlings to solutions with water potential values of −3 to −11 bars effected an immediate cessation of leaf elongation with growth resumption after several minutes or hours. Latent periods were increased and growth resumption rates were decreased as water potential values of nutrient solutions were lowered. In unstressed transpiring seedlings, water potential and osmotic potential values of leaf basal tissues were usually −6 to −8 bars, and −12 to −14 bars, respectively. These tissues began to adjust osmotically when exposed to any of the osmotic solutions, and hourly reductions of 1 to 2 bars in both water potential and osmotic potential values usually occurred for the first 2 to 4 hours, but reduction rates thereafter were lower. When seedlings were exposed to solutions with water potential values lower than those of the leaf basal tissues, growth resumed about the time water potential values of those tissues fell to that of the nutrient solution. After 1 to 3 days of seedling exposure to solutions with different water potential values, cumulative leaf elongation was reduced as the water potential values of the root medium were lowered. Reductions in water potential and osmotic potential values of tissues in leaf basal regions paralleled growth reductions, but turgor value was largely unaffected by stress. In contrast, water potential, osmotic potential, and turgor values of leaf blades were usually changed slightly regardless of the degree and duration of stress, and blade water potential values were always higher than water potential values of the basally located cells. It is hypothesized that blades have high water potential values and are generally unresponsive to stress because water in most of the mesophyll cells in this area does not exchange readily with water present in the transpiration stream.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

Response of carbon dioxide fixation to water stress: parallel measurements on isolated chloroplasts and intact spinach leaves

Application of water stress to isolated spinach (Spinacia oleracea) chloroplasts by redutcion of the osmotic potentials of CO₂ fixation media below −6 to −8 bars resulted in decreased rates of fixation regardless of solute composition. A decrease in CO₂ fixation rate of isolated chloroplasts was also found when leaves were dehydrated in air prior to chloroplast isolation. An inverse response of CO₂ fixation to osmotic potential of the fixation medium was found with chloroplasts isolated from dehydrated leaves—namely, fixation rate was inhibited at −8 bars, compared with −16 or −24 bars.     

Osmotic adjustment in leaves of sorghum in response to water deficits

The relationships among the total water potential, osmotic potential, turgor potential, and relative water content were determined for leaves of sorghum (Sorghum bicolor [L.] Moench cvs. `RS 610' and `Shallu') with three different histories of water stress. Plants were adequately watered (control), or the soil was allowed to dry slowly until the predawn leaf water potential reached either −0.4 megapascal (MPa) (treatment A) or −1.6 MPa (treatment B). Severe soil and plant water deficits developed sooner after cessation of watering in `Shallu' than in `RS 610', but no significant differences in osmotic adjustment or tissue water relations were observed between the two cultivars. In both cultivars, the stress treatments altered the relationship between leaf water potential and relative water content, resulting in the previously stressed plants maintaining higher tissue water contents than control plants at the same leaf water potential. The osmotic potential at full turgor in the control sorghum was −0.7 MPa: stress pretreatment significantly lowered the osmotic potential to −1.1 and −1.6 MPa in stress treatments A and B, respectively. As a result of this osmotic adjustment, leaf turgor potentials at a given value of leaf water potential exceeded those of the control plants by 0.15 to 0.30 MPa in treatment A and by 0.5 to 0.65 MPa in treatment B. However, zero turgor potential occurred at approximately the same value of relative water content (94%) irrespective of previous stress history. From the relationship between turgor potential and relative water content there was an approximate doubling of the volumetric elastic modulus, i.e. a halving of tissue elasticity, as a result of stress preconditioning. The influence of stress preconditioning on the moisture release curve is discussed.     

Evaluation of water stress control with polyethylene glycols by analysis of guttation

The water relations of pepper plants (Capsicum frutescens L.) under conditions conducive to guttation were studied to evaluate the control of plant water stress with polyethylene glycols. The addition of polyethylene glycol 6000 to the nutrient solution resulted in water relations similar to those expected in soil at the same water potentials. Specifically, xylem pressure potential in the root and leaf became more negative during a 24-hour treatment period, while osmotic potential of the root xylem sap remained constant. The decrease in pressure potential was closely correlated with the decrease in osmotic potential of the nutrient solution. In contrast, the addition of polyethylene glycol 400 to the nutrient medium resulted in a reduction of osmotic potential in the root xylem sap; this osmotic adjustment in the xylem was large enough to establish an osmotic gradient for entry of water and cause guttation at a nutrient solution osmotic potential of −4.8 bars. Pressure potential in the root and leaf xylem became negative only at nutrient solution osmotic potentials lower than −4.8 bars. About half of the xylem osmotic adjustment in the presence of polyethylene glycol 400 was caused by increased accumulation of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the root xylem. These studies indicate that larger polyethylene glycol molecules such as polyethylene glycol 6000 are more useful for simulating soil water stress than smaller molecules such as polyethylene glycol 400.     

Proline accumulation and the adaptation of cultured plant cells to water stress

The transfer of cultured tomato cells (Lycopersicon esculentum cv VFNT-Cherry) to a low water potential environment resulted in an increased dry weight to fresh weight ratio accompanied by a rapid accumulation of proline. Proline content continued to increase as osmotic adjustment and growth occurred. The initial increase in proline concentration was accompanied by a drop in turgor. However, proline levels continued to increase with a gain in turgor during osmotic adjustment. Thus, the accumulation of proline depended not only on cell water potential, or on the initial loss of turgor but more closely on cell osmotic potential. The ultimate level of proline depended on the level of adaptation. Proline levels remained high after more than 100 cell generations in low water potential media, but declined rapidly after transfer to media with a less negative water potential. Addition of exogenous proline to the medium during water stress and during osmotic downshock alleviated the normally resulting inhibition of growth. The results suggest a positive role for proline accumulation in adaptation of cells to changing external water potentials.     

Solute Accumulation in Tobacco Cells Adapted to NaCl

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCl (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na⁺ and Cl⁻ as principal solutes for this adjustment. Although the intracellular concentrations of Na⁺ and Cl⁻ correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na⁺ and Cl⁻ did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na⁺ and Cl⁻. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar NaCl and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na⁺ and Cl⁻ are principal components of osmotic adjustment, organic solutes also may make significant contributions.     

Carbon balance and water relations of sorghum exposed to salt and water stress

The daily (24 hour) changes in carbon balance, water loss, and leaf area of whole sorghum plants (Sorghum bicolor L. Moench, cv BTX616) were measured under controlled environment conditions typical of warm, humid, sunny days. Plants were either (a) irrigated frequently with nutrient solution (osmotic potential −0.08 kilojoules per kilogram = −0.8 bar), (b) not irrigated for 15 days, (c) irrigated frequently with moderately saline nutrient (80 millimoles NaCl + 20 millimoles CaCl₂·2H₂O per kilogram water, osmotic potential −0.56 kilojoules per kilogram), or (d) preirrigated with saline nutrient and then not irrigated for 22 days.

Under frequent irrigation, salt reduced leaf expansion and carbon gain, but water use efficiency was increased since the water loss rate was reduced more than the carbon gain. Water stress developed more slowly in the salinized plants and they were able to adjust osmotically by a greater amount. Leaf expansion and carbon gain continued down to lower leaf water potentials.

Some additional metabolic cost associated with salt stress was detected, but under water stress this was balanced by the reduced cost of storing photosynthate rather than converting it to new biomass. Reirrigation produced a burst of respiration associated with renewed synthesis of biomass from stored photosynthate.

It is concluded that although irrigation of sorghum with moderately saline water inhibits plant growth in comparison with irrigation with nonsaline water, it also inhibits water loss and allows a greater degree of osmotic adjustment, so that the plants are able to continue growing longer and reach lower leaf water potentials between irrigations.

     

Detrimental effect of rust infection on the water relations of bean

Bean plants (Phaseolus vulgaris L.) infected with the rust Uromyces phaseoli became unusually susceptible to drought as sporulation occurred. Under the conditions used (1,300 ft-c, 27 C, and 55% relative humidity) such plants wilted at soil water potentials greater than −1 bar, whereas healthy plants did not wilt until the soil water potential fell below −3.4 bars. Determinations of leaf water and osmotic potentials showed that an alteration in leaf osmotic potential was not responsible for the wilting of diseased plants. When diffusive resistance was measured as a function of decreasing leaf water content, the resistance of healthy leaves increased to 50 sec cm⁻¹ by the time relative water content decreased to 70%, whereas the resistance of diseased leaves remained less than 8 sec cm⁻¹ down to 50% relative water content. Apparently, water vapor loss through cuticle damaged by the sporulation process, together with the reduction in root to shoot ratio which occurs in diseased plants, upset the water economy of the diseased plant under mild drought conditions.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Rice Chalky Ring Formation Caused by Temporal Reduction in Starch Biosynthesis during Osmotic Adjustment under Foehn-Induced Dry Wind

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s⁻¹) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.     

Endosperm cell division in maize kernels cultured at three levels of water potential

The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of −1.1, −1.6, or −2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of −1.6 or −2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control (−1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the −2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at −1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two −2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors.     

Extracellular polysaccharides and proteins of tobacco cell cultures and changes in composition associated with growth-limiting adaptation to water and saline stress

The chemical composition of extracellular polymers released by cells of tobacco (Nicotiana tabacum L. cv W38) adapted to a medium containing 30% polyethylene glycol 8000 (−28 bar) or 428 millimolar NaCl (−23 bar) was compared to the composition of those released by unadapted cells. Unadapted cells released uronic acid-rich material of high molecular weight, arabinogalactan-proteins, low molecular weight fragments of hemicellulosic polysaccharides, and a small amount of protein. Cells adapted to grow in medium containing NaCl released arabinogalactan and large amounts of protein but not the uronic acid-rich material, and cells adapted to grow in polyethylene glycol released only small amounts of an arabinogalactan of much lower molecular weight and some protein. Secretion of all material was nearly blocked by polyethylene glycol, but when cells were transferred to a medium containing iso-osmolar mannitol, they again released extracellular polymers at rates similar to those of unadapted cells. Like cells adapted to NaCl, however, these cells released arabinogalactan and large amounts of protein but only small amounts of the uronic acid-rich material. Media of NaCl-adapted cells were enriched in 40, 29, and 11 kilodalton polypeptides. CaCl₂ extracted the 40 and 11 kilodalton polypeptides from walls of unadapted cells, but the 29 kilodalton polypeptide was found only in the medium of the NaCl-adapted cells. Accumulation of low molecular weight polysaccharide fragments in the medium was also substantially reduced in both NaCl- and polyethylene glycol-adapted cells, and specifically, the material was composed of lower proportions of xyloglucan fragments. Our results indicate that adaptation to saline or water stress results in inhibition of both the hydrolysis of hemicellulosic xyloglucan and release of uronic acid-rich material into the culture medium.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Growth-induced Water Potentials in Plant Cells and Tissues

A physical analysis of water movement through elongating soybean (Glycine max L. Merr.) hypocotyls was made to determine why significant water potentials persist in growing tissues even though the external water potentials were zero and transpiration is virtually zero. The analysis was based on a water transport theory modified for growth and assumed that water for growing cells would move through and along the cells in proportion to the conductivity of the various pathways.

Water potentials calculated for individual cells were nearly in local equilibrium with the water potentials of the immediate cell surroundings during growth. However, water potentials calculated for growing tissue were 1.2 to 3.3 bars below the water potential of the vascular supply in those cells farthest from the xylem. Only cells closest to the xylem had water potentials close to that of the vascular supply. Gradients in water potential were steepest close to the xylem because all of the growth-sustaining water had to move through this part of the tissue. Average water potentials calculated for the entire growing region were −0.9 to −2.2 bars depending on the tissue diffusivity.

For comparison with the calculations, average water potentials were measured in elongating soybean hypocotyls using isopiestic thermocouple psychrometers for intact and excised tissue. In plants having virtually no transpiration and growing in Vermiculite with a water potential of −0.1 bar, rapidly growing hypocotyl tissue had water potentials of −1.7 to −2.1 bars when intact and −2.5 bars when excised. In mature, nongrowing hypocotyl tissue, average water potentials were −0.4 bar regardless of whether the tissue was intact or excised.

The close correspondence between predicted and measured water potentials in growing tissue indicates that significant gradients in water potential are required to move growth-associated water through and around cells over macroscopic distances. The presence of such gradients during growth indicates that cells must have different cell wall and/or osmotic properties at different positions in the tissue in order for organized growth to occur. The mathematical development used in this study represents the philosophy that would have to be followed for the application of contemporary growth theory when significant tissue water potential gradients are present.