Direct Demonstration of a Growth-Induced Water Potential Gradient

When transpiration is negligible, water potentials in growing tissues are less than those in mature tissues and have been predicted to form gradients that move water into the enlarging cells. To determine directly whether the gradients exist, we measured water potentials along the radius of stems of intact soybean (Glycine max [L.] Merr.) seedlings growing in vermiculite in a water-saturated atmosphere. The measurements were made in individual cells by first determining the turgor with a miniature pressure probe, then determining the osmotic potential of solution from the same cell, and finally summing the two potentials. The osmotic potentials were corrected for sample mixing in the probe. The measurements were checked with a thermocouple psychrometer that gave average tissue water potentials. In the elongating region, the water potential was highest near the xylem and lowest near the epidermis and in the center of the pith. In the basal, more mature region of the same stems, water potentials were near zero next to the xylem and throughout the tissue. These basal potentials reflected mostly the potential of the xylem, which extended into the elongating tissues. Thus, the high basal potential confirmed the high potential near the xylem in the elongating tissues. The psychrometer measurements for each tissue gave average potentials that agreed with the average of the cell potentials from the pressure probe. We conclude that a radial gradient was present in the elongating region that formed a water potential field in three dimensions around the xylem and that confirmed the predictions of Molz and Boyer (F.J. Molz and J.S. Boyer [1978] Plant Physiol 62: 423-429).     

Root Growth Maintenance at Low Water Potentials (Increased Activity of Xyloglucan Endotransglycosylase and Its Possible Regulation by Abscisic Acid)

Previous work suggested that an increase in cell wall-loosening contributes to the maintenance of maize (Zea mays L.) primary root elongation at low water potentials ([psi]w). It was also shown that root elongation at low [psi]w requires increased levels of abscisic acid (ABA). In this study we investigated the effects of low [psi]w and ABA status on xyloglucan endotransglycosylase (XET) activity in the root elongation zone. XET is believed to contribute to wall-loosening by reversibly cleaving xyloglucan molecules that tether cellulose microfibrils. The activity of XET per unit fresh weight in the apical 10 mm (encompassing the elongation zone) was constant at high [psi]w but increased by more than 2-fold at a [psi]w of -1.6 MPa. Treatment with fluridone to decrease ABA accumulation greatly delayed the increase in activity at low [psi]w. This effect was largely overcome when internal ABA levels were restored by exogenous application. Spatial distribution studies showed that XET activity was increased in the apical 6 mm at low [psi]w whether expressed per unit fresh weight, total soluble protein, or cell wall dry weight, corresponding to the region of continued elongation. Treatment with fluridone progressively inhibited the increase in activity with distance from the apex, correlating with the pattern of inhibition of elongation. Added ABA partly restored activity at all positions. The increase in XET activity at low [psi]w was due to maintenance of the rate of deposition of activity despite decreased deposition of wall material. The loss of activity associated with decreased ABA was due to inhibition of the deposition of activity. The results demonstrate that increased XET activity is associated with maintenance of root elongation at low [psi]w and that this response requires increased ABA.     

Growth of the Maize Primary Root at Low Water Potentials : II. Role of Growth and Deposition of Hexose and Potassium in Osmotic Adjustment

Primary roots of maize (Zea mays L. cv WF9 × Mo17) seedlings growing in vermiculite at various water potentials exhibited substantial osmotic adjustment in the growing region. We have assessed quantitatively whether the osmotic adjustment was attributable to increased net solute deposition rates or to slower rates of water deposition associated with reduced volume expansion. Spatial distributions of total osmotica, soluble carbohydrates, potassium, and water were combined with published growth velocity distributions to calculate deposition rate profiles using the continuity equation. Low water potentials had no effect on the rate of total osmoticum deposition per unit length close to the apex, and caused decreased deposition rates in basal regions. However, rates of water deposition decreased more than osmoticum deposition. Consequently, osmoticum deposition rates per unit water volume were increased near the apex and osmotic potentials were lower throughout the growing region. Because the stressed roots were thinner, osmotic adjustment occurred without osmoticum accumulation per unit length. The effects of low water potential on hexose deposition were similar to those for total osmotica, and hexose made a major contribution to the osmotic adjustment in middle and basal regions. In contrast, potassium deposition decreased at low water potentials in close parallel with water deposition, and increases in potassium concentration were small. The results show that growth of the maize primary root at low water potentials involves a complex pattern of morphogenic and metabolic events. Although osmotic adjustment is largely the result of a greater inhibition of volume expansion and water deposition than solute deposition, the contrasting behavior of hexose and potassium deposition indicates that the adjustment is a highly regulated process.     

Potassium Loss from Stomatal Guard Cells at Low Water Potentials

The potassium content of guard cells and the resistance to viscousflow of air through the leaf were determined in sunflower (Helianthusannuus) subjected to low leaf water potentials under illuminatedconditions. In intact plants desiccated slowly by withholdingwater from the soil, large losses in guard cell K occurred asleaf water potentials decreased. Leaf viscous resistance increased,indicating stomatal closure. Similar results were obtained whendetached leaf segments were desiccated rapidly. Upon rehydrationof leaves, no stomatal opening was observed initially, despiteleaf water potentials at predesiccated levels. After severalhours, however, re-entry of K occurred and stomata became fullyopen. Turgid leaf segments floated on an ABA solution showedlosses of guard cell K and closure of stomata as rapidly andcompletely as those brought about by desiccation. It is concludedthat stomatal closure at low water potentials under illuminatedconditions is not controlled solely by water loss from the tissuebut involves the loss of osmoticum from the guard cells as well.This in turn decreases the turgor difference between the guardcells and the surrounding cells, and closing occurs.     

Proline Accumulation in Maize (Zea mays L.) Primary Roots at Low Water Potentials (I. Requirement for Increased Levels of Abscisic Acid)

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.     

Translatable RNA Populations Associated with Maintenance of Primary Root Elongation and Inhibition of Mesocotyl Elongation by Abscisic Acid in Maize Seedlings at Low Water Potentials

Previous work indicated that accumulation of abscisic acid (ABA) acts differentially to maintain elongation of the primary root and inhibit elongation of the mesocotyl of maize (Zea mays L.) seedlings at low water potentials ([psi]w). Subsequent results indicated specific locations in the elongation zones where elongation is maintained, inhibited, or unaffected by endogenous ABA at low [psi]w. This information was utilized in this study to identify in vitro translation products of RNA associated with the maintenance or inhibition of elongation in the primary root and mesocotyl, respectively, by endogenous ABA at low [psi]w. The results distinguished products associated specifically with the elongation responses from those nonspecifically associated with ABA accumulation or low [psi]w, as well as normal cell development and maturation. In the primary root, the maintenance of elongation at low [psi]w by ABA was associated with the maintenance of expression of three products that were also expressed during elongation at high [psi]w, the expression of a novel product, and the suppression of two products. In the mesocotyl, the inhibition of elongation by ABA after transplanting to low [psi]w was associated with the induction of a novel translation product. However, the induction of this product, as well as accumulation of ABA and inhibition of elongation, occurred without a decline in tissue water content. The results demonstrate the necessity of examining the association of gene expression with elongation responses to low [psi]w with a high degree of spatial resolution.     

Photophosphorylation in Attached Leaves of Helianthus annuus at Low Water Potentials

The in situ response of photophosphorylation and coupling factor activity to low leaf water potential (ψ_L) was investigated using kinetic spectroscopy to measure the flash-induced electrochromic absorption change in attached sunflower (Helianthus annuus L. cv IS894) leaves. The electrochromic change is caused by the formation of an electric potential across the thylakoid membrane associated with proton uptake. Since depolarization of the thylakoid membrane following flash excitation is normally dominated by proton efflux through the coupling factor during ATP formation, this measurement can provide direct information about the catalytic activity of the coupling factor. Under low ψ_L conditions in which a clear nonstomatal limitation of net photosynthesis could be demonstrated, we found a strong inhibition of coupling factor activity in dark-adapted leaves which was probably caused by an increase in the energetic threshold for the activation of the enzyme at low ψ_L. While this result supported earlier in vitro findings, we further discovered that the light-dependent reduction of coupling factor reversed any observable effect of low ψ_L on the energetics of activation or on photophosphorylation competence. Furthermore, coupling factor was reduced, even in severely droughted sunflower, almost immediately upon illumination. Based on these measurements, we conclude that the nonstomatal limitation of photosynthesis observed by us and others in droughted plants cannot be explained by impaired coupling factor activity.     

Chloroplast Response to Low Leaf Water Potentials: II. Role of Osmotic Potential

Electron transport in chloroplasts isolated from desiccated sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves was compared with electron transport in sunflower chloroplasts in sorbitol-containing media having various osmotic potentials. In media having low osmotic potentials and dichloroindophenol as electron acceptor, the activity for electron transport was inhibited, but the inhibition was much less than that due to comparable desiccation in vivo. The inhibition at low osmotic potentials was rapidly reversed by returning the chloroplasts to media having high osmotic potentials, but the activity of chloroplasts from desiccated tissue showed no reversal when the chloroplasts were placed in media having high osmotic potentials. Nevertheless, the inhibition of chloroplast activity due to desiccation in vivo was basically reversible, because chloroplasts recovered quickly when they were rehydrated in vivo. The large differences between desiccation in vivo and exposure to low osmotic potential in vivo indicate that osmotic solutions did not reproduce the effects of tissue desiccation. It is concluded that decreases in the Gibbs free energy of water due to decreased osmotic potentials probably have only a small effect on electron transport in chloroplasts from desiccated tissue and do not account for the major effects of leaf desiccation on electron transport.     

Resistance to Water Transport in Shoots of Vitis vinifera L. : Relation to Growth at Low Water Potential

Apparent resistances to water transport in the liquid phase were determined from measurements of soil, root, basal shoot internode, shoot apex, and leaf water potentials and water flux in Vitis vinifera (cv White Riesling) during soil drying. Predawn water potential differences (ΔΨ) in the shoots accounted for 20% of the total ΔΨ between the soil and the shoot apex when plants were well-watered but increased to about 90% when shoot growth ceased. The ΔΨ from soil to root was essentially constant during this period. At low water potential, the ΔΨ in the shoot was persistent when transpiration was low (predawn) or completely prevented (plant bagging). The apparent hydraulic resistance between the basal shoot internode and most rapidly expanding leaf (or shoot apex) increased several-fold when water was withheld. Leaf and internode expansion both exhibited high sensitivity to increasing hydraulic resistance. Measurements of pneumatic resistance to air flow through frozen internode segments indicated progressive vapor-filling of vessels as soil drying progressed. From these observations and others in the literature, it was suggested that embolization may be a common occurrence and play an important role in the inhibition of shoot growth at moderate water deficits.     

Internal CO(2) Measured Directly in Leaves : Abscisic Acid and Low Leaf Water Potential Cause Opposing Effects

Observations of nonuniform photosynthesis across leaves cast doubt on internal CO₂ partial pressures (p_i) calculated on the assumption of uniformity and can lead to incorrect conclusions about the stomatal control of photosynthesis. The problem can be avoided by measuring p_i directly because the assumptions of uniformity are not necessary. We therefore developed a method that allowed p_i to be measured continuously in situ for days at a time under growth conditions and used it to investigate intact leaves of sunflower (Helianthus annuus L.), soybean (Glycine max L. Merr.), and bush bean (Phaseolus vulgaris L.) subjected to high or low leaf water potentials (ψ_w) or high concentrations of abscisic acid (ABA). The leaves maintained a relatively constant differential (Δp) between ambient CO₂ and measured p_i throughout the light period when water was supplied. When water was withheld, ψ_w decreased and the stomata began to close, but measured p_i increased until the leaf reached a ψ_w of −1.76 (bush bean), −2.12 (sunflower) or −3.10 (soybean) megapascals, at which point Δp = 0. The increasing p_i indicated that stomata did not inhibit CO₂ uptake and a Δp of zero indicated that CO₂ uptake became zero despite the high availability of CO₂ inside the leaf. In contrast, when sunflower leaves at high ψ_w were treated with ABA, p_i did not increase and instead decreased rapidly and steadily for up to 8 hours even as ψ_w increased, as expected if ABA treatment primarily affected stomatal conductance. The accumulating CO₂ at low ψ_w and contrasting response to ABA indicates that photosynthetic biochemistry limited photosynthesis at low ψ_w but not at high ABA.     

Starch and the Control of Kernel Number in Maize at Low Water Potentials

After reproduction is initiated in plants, subsequent reproductive development is sometimes interrupted, which decreases the final number of seeds and fruits. We subjected maize (Zea mays L.) to low water potentials (psi(w)) that frequently cause this kind of failure. We observed metabolite pools and enzyme activities in the developing ovaries while we manipulated the sugar stream by feeding sucrose (Suc) to the stems. Low psi(w) imposed for 5 d around pollination allowed embryos to form, but abortion occurred and kernel number decreased markedly. The ovary contained starch that nearly disappeared during this abortion. Analyses showed that all of the intermediates in starch synthesis were depleted. However, when labeled Suc was fed to the stems, label arrived at the ovaries. Solute accumulated and caused osmotic adjustment. Suc accumulated, but other intermediates did not, showing that a partial block in starch synthesis occurred at the first step in Suc utilization. This step was mediated by invertase, which had low activity. Because of the block, Suc feeding only partially prevented starch disappearance and abortion. These results indicate that young embryos abort when the sugar stream is interrupted sufficiently to deplete starch during early ovary development, and this abortion results in a loss of mature seeds and fruits. At low psi(w), maintaining the sugar stream partially prevented the abortion, but invertase regulated the synthesis of ovary starch and partially prevented full recovery.     

CO2 and Water Vapor Exchange across Leaf Cuticle (Epidermis) at Various Water Potentials

Cuticular properties affect the gas exchange of leaves, but little is known about how much CO2 and water vapor cross the cuticular barrier or whether low water potentials affect the process. Therefore, we measured the cuticular conductances for CO2 and water vapor in grape (Vitis vinifera L.) leaves having various water potentials. The lower leaf surface was sealed to force all gas exchange through the upper surface, which was stoma-free. In this condition both gases passed through the cuticle, and the CO2 conductance could be directly determined from the internal mole fraction of CO2 near the compensation point, the external mole fraction of CO2, and the CO2 flux. The cuticle allowed small amounts of CO2 and water vapor to pass through, indicating that gas exchange occurs in grape leaves no matter how tightly the stomata are closed. However, the CO2 conductance was only 5.7% of that for water vapor. This discrimination against CO2 markedly affected calculations of the mole fraction of CO2 in leaves as stomatal apertures decreased. When the leaf dehydrated, the cuticular conductance to water vapor decreased, and transpiration and assimilation diminished. This dehydration effect was largest when turgor decreased, which suggests that cuticular gas exchange may have been influenced by epidermal stretching.     

Chloroplast Response to Low Leaf Water Potentials: III. Differing Inhibition of Electron Transport and Photophosphorylation

Cyclic and noncyclic photophosphorylation and electron transport by photosystem 1, photosystem 2, and from water to methyl viologen (“whole chain”) were studied in chloroplasts isolated from sunflower (Helianthus annus L. var Russian Mammoth) leaves that had been desiccated to varying degrees. Electron transport showed considerable inhibition at leaf water potentials of −9 bars when the chloroplasts were exposed to an uncoupler in vitro, and it continued to decline in activity as leaf water potentials decreased. Electron transport by photosystem 2 and coupled electron transport by photosystem 1 and the whole chain were unaffected at leaf water potentials of −10 to −11 bars but became progressively inhibited between leaf water potentials of −11 and −17 bars. A low, stable activity remained at leaf water potentials below −17 bars. In contrast, both types of photophosphorylation were unaffected by leaf water potentials of −10 to −11 bars, but then ultimately became zero at leaf water potentials of −17 bars. Although the chloroplasts isolated from the desiccated leaves were coupled at leaf water potentials of −11 to −12 bars, they became progressively uncoupled as leaf water potentials decreased to −17 bars. Abscisic acid and ribonuclease had no effect on chloroplast photophosphorylation. The results are generally consistent with the idea that chloroplast activity begins to decrease at the same leaf water potentials that cause stomatal closure in sunflower leaves and that chloroplast electron transport begins to limit photosynthesis at leaf water potentials below about −11 bars. However, it suggests that, during severe desiccation, the limitation may shift from electron transport to photophosphorylation.     

Root Growth and Oxygen Relations at Low Water Potentials. Impact of Oxygen Availability in Polyethylene Glycol Solutions

Polyethylene glycol (PEG), which is often used to impose low water potentials (ψ_w) in solution culture, decreases O₂ movement by increasing solution viscosity. We investigated whether this property causes O₂ deficiency that affects the elongation or metabolism of maize (Zea mays L.) primary roots. Seedlings grown in vigorously aerated PEG solutions at ambient solution O₂ partial pressure (pO₂) had decreased steady-state root elongation rates, increased root-tip alanine concentrations, and decreased root-tip proline concentrations relative to seedlings grown in PEG solutions of above-ambient pO₂ (alanine and proline accumulation are responses to hypoxia and low ψ_w, respectively). Measurements of root pO₂ were made using an O₂ microsensor to ensure that increased solution pO₂ did not increase root pO₂ above physiological levels. In oxygenated PEG solutions that gave maximal root elongation rates, root pO₂ was similar to or less than (depending on depth in the tissue) pO₂ of roots growing in vermiculite at the same ψ_w. Even without PEG, high solution pO₂ was necessary to raise root pO₂ to the levels found in vermiculite-grown roots. Vermiculite was used for comparison because it has large air spaces that allow free movement of O₂ to the root surface. The results show that supplemental oxygenation is required to avoid hypoxia in PEG solutions. Also, the data suggest that the O₂ demand of the root elongation zone may be greater at low relative to high ψ_w, compounding the effect of PEG on O₂ supply. Under O₂-sufficient conditions root elongation was substantially less sensitive to the low ψ_w imposed by PEG than that imposed by dry vermiculite.     

Proline Metabolism and Transport in Maize Seedlings at Low Water Potential

The growing zone of maize seedling primary roots accumulatesproline at low water potential. Endosperm removal and excisionof root tips rapidly decreased the proline pool and greatlyreduced proline accumulation in root tips at low water potential.Proline accumulation was not restored by exogenous amino acids.Labelling root tips with [¹⁴C]glutamate and [¹⁴C]proline showedthat the rate of proline utilization (oxidation and proteinsynthesis) exceeded the rate of biosynthesis by five-fold athigh and low water potentials. This explains the reduction inthe proline pool following root and endosperm excision and theinability to accumulate proline at low water potential. Theendosperm is therefore the source of the proline that accumulatesin the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [¹⁴C]Prolinewas transported from the scutellum to other parts of the seedlingand reached the highest concentration in the root tip. Less[¹⁴C]proline was transported at low water potential but becauseof the lower rate of protein synthesis and oxidation, more accumulatedas proline in the root tip. Despite the low biosynthesis capacityof the roots, the extent of proline accumulation in relationto water potential is precisely controlled by transport andutilization rate.     

134 Cryopreservation of Chlamydomonas Reinhardtii (Chlorophyceae): A Cause of Low Viability at High Cell Density

Cryopreservation provides a convenient method for long term storage of living organisms. Current protocols allow the successful cryopreservation of a wide range of algae, although many strains remain recalcitrant to cryopreservation. Chlamydomonas reinhardtii , a species utilized in many molecular and biochemical studies, survives cryopreservation best at low cell density. We show that reduced viability at higher cell densities is caused by the accumulation of a substance released from C. reinhardtii into the culture medium during cryopreservation. A mutant strain of C. reinhardtii (cw10) with a greatly reduced cell wall did not release a substance inhibitory to wild type or cw10 C. reinhardtii during cryopreservation, and could be cryopreserved with the same viability regardless of cell density. The inhibitory substance is small (mw<1300), polar, heat-stable and organic. Chlamydomonas moewusii Gerloff and Chlamydomonas zebra Korschikov ex Pascher both produce substances that reduce the viability of cryopreserved C. reinhardtii . However, neither is affected by the inhibitory substance produced by themselves or C. rienhardtii. Pandorina morum (Müller) Bory and Volvox carteri f. nagariensis Iyengar are colonial Volvocalean algae related to C. reinhardtii that cannot be successfully cryopreserved. They both generate substances that inhibit C. reinhardtii during cryopreservation. The identification of the substance inhibitory to C. reinhardtii during cryopreservation should explain why this alga cryopreserves best at a low cell density, and may lead to protocols that facilitate the more successful cryopreservation of C. reinhardtii and related algae.     

Photosynthetic Oxygen Evolution at Low Water Potential in Leaf Discs Lacking an Epidermis

Land plants encountering low water potentials (low _w) closetheir stomata, restricting CO₂ entry and potentially photosynthesis.To determine the impact of stomatal closure, photosyntheticO₂ evolution was investigated in leaf discs from sunflower (Helianthusannuus L.) plants after removing the lower epidermis at low_w. Wounding was minimal as evidenced by O₂ evolution nearlyas rapid as that in intact discs. O₂ evolution was maximal in1 % CO₂ in the peeled discs and was markedly inhibited when_w was below –1·1 MPa. CO₂ entered readily at all_w, as demonstrated by varying the CO₂ concentration. Resultswere the same whether the epidermis was removed before or afterlow _w was imposed. Due to the lack of an epidermis and readymovement of CO₂ through the mesophyll, the loss in O₂ evolvingactivity was attributed entirely to photosynthetic metabolism.Intact leaf discs showed a similar loss in activity when measuredat a CO₂ concentration of 5 %, which supported maximum O₂ evolutionat low _w. In 1 % CO₂, however, O₂ evolution at low _w was belowthe maximum, presumably because stomatal closure restrictedCO₂ uptake. The inhibition was larger than in peeled discs at_w between –1 and –1·5 MPa but became thesame as in peeled discs at lower _w. Therefore, as photosynthesisbegan to be inhibited by metabolism at low _w, stomatal closureadded to the inhibition. As _w became more negative, the inhibitionbecame entirely metabolic.     

Growth of Aeromonas hydrophila at Low Concentrations of Substrates Added to Tap Water

The ability of an Aeromonas hydrophila isolate obtained from filtered river water to grow at low substrate concentrations was studied in batch experiments with tap water supplied with low concentrations of substrates. Growth was assessed by colony count determinations. The isolate only multiplied in the used tap water (2 to 3 mg of dissolved organic carbon per liter) after the addition of a small amount of an assimilable carbon compound. d-Glucose especially caused growth of the organism even at initial concentrations below 10 mug of C per liter. At initial glucose concentrations below the K(s) value (12 mug of C per liter), generation times and yield (colony-forming units per milligram of substrate-C) were nonlinear with 1/initial glucose concentrations and initial glucose concentrations, respectively. From these observations, the maintenance coefficient m was calculated (m = 0.015 mg of glucose per mg [dry wt] per h at 12 degrees C). At initial concentrations below the K(s) value of starch (73 mug of C per liter), no growth was observed, but complete use of starch occurred in these situations after the addition of 10 mug of glucose-C per liter. The results of this study show that information of ecological significance may be obtained by very simple batch experiments. Moreover, the isolate studied may be used in growth experiments to assess the maximum concentration of glucose which might be present in water, particularly tap water.