Water Relations of Pachysandra Leaves during Freezing and Thawing : Evidence for a Negative Pressure Potential Alleviating Freeze-Dehydration Stress

The evergreen herb Pachysandra terminalis becomes moderately frost-hardy in winter. The water relations of its frost-hardy leaves were studied during a freeze-thaw cycle. Leaf water potentials, measured by psychrometry at subfreezing temperatures, were identical with those of ice, indicating equilibrium freezing. Microscopic observations showed extracellular freezing of tissue water. As evidenced by thermal analysis, the freezing process starts with the crystallization of a minor volume which was identified as apoplasmic water. The following long-lasting exotherm indicated slow export of water from the protoplasts driven by extracellular crystallization. In partially frozen leaves, the fractions of liquid water were measured at several subfreezing temperatures by nuclear magnetic resonance spectroscopy. They were consistently greater than those calculated from the osmotic potentials of cellular fluid, and the differences increased with decreasing temperature. About 50% of the differences could be abolished by freeze-killing of the leaf and was thus ascribed to the effect of a (negative) pressure reinforcing the osmotic potential. The persistent part of the differences may have reflected a matric component. At −7°C, the absolute values of both potentials were −1.7 megapascals each. The water relations of Pachysandra leaves clearly indicate nonideal equilibrium freezing where negative pressures and matric potentials contribute to the leaf water potential and thus alleviate freeze-dehydration of the tissue.     

Relationship of water potential to growth of leaves

A thermocouple psychrometer that measures water potentials of intact leaves was used to study the water potentials at which leaves grow. Water potentials and water uptake during recovery from water deficits were measured simultaneously with leaves of sunflower (Helianthus annuus L.), tomato (Lycopersicon esculentum Mill.), papaya (Carica papaya L.), and Abutilon striatum Dickson. Recovery occurred in 2 phases. The first was associated with elimination of water deficits; the second with cell enlargement. The second phase was characterized by a steady rate of water uptake and a relatively constant leaf water potential. Enlargement was 70% irreversible and could be inhibited by puromycin and actinomycin D. During this time, leaves growing with their petioles in contact with pure water remained at a water potential of —1.5 to —2.5 bars regardless of the length of the experiment. It was not possible to obtain growing leaf tissue with a water potential of zero. It was concluded that leaves are not in equilibrium with the potential of the water which is absorbed during growth. The nonequilibrium is brought about by a resistance to water flow which requires a potential difference of 1.5 to 2.5 bars in order to supply water at the rate necessary for maximum growth.

Leaf growth occurred in sunflower only when leaf water potentials were above —3.5 bars. Sunflower leaves therefore require a minimum turgor for enlargement, in this instance equivalent to a turgor of about 6.5 bars. The high water potentials required for growth favored rapid leaf growth at night and reduced growth during the day.

     

Matric Water Potential of Leaf Tissue--Measurement and Significance

A thermocouple psychrometer method, previously described foruse in determining tissue and sap water potentials, has beenadapted for determining matric potentials. Matric values ranging from approximately zero to –10 x10⁵ Pa were observed with wilting wheat leaves. Mean valuesof total tissue water potential and of its solute (osmotic),matric, and cell wall pressure components were –16.6,–18.3, –30 and +4.7 Pa x 10⁵, respectively. Matric potentials are often ignored in investigations of plantwater relations. This practice is shown to result in underestimationof wall pressures (in this case by a mean of 64 per cent) andsometimes in spuriously negative values.     

Leaf water potentials measured with a pressure chamber

Leaf water potentials were estimated from the sum of the balancing pressure measured with a pressure chamber and the osmotic potential of the xylem sap in leafy shoots or leaves. When leaf water potentials in yew, rhododendron, and sunflower were compared with those measured with a thermocouple psychrometer known to indicate accurate values of leaf water potential, determinations were within ± 2 bars of the psychrometer measurements with sunflower and yew. In rhododendron. water potentials measured with the pressure chamber plus xylem sap were 2.5 bars less negative to 4 bars more negative than psychrometer measurements.

The discrepancies in the rhododendron measurements could be attributed, at least in part, to the filling of tissues other than xylem with xylem sap during measurements with the pressure chamber. It was concluded that, although stem characteristics may affect the measurements, pressure chamber determinations were sufficiently close to psychrometer measurements that the pressure chamber may be used for relative measurements of leaf water potentials, especially in sunflower and yew. For accurate determinations of leaf water potential, however, pressure chamber measurements must be calibrated with a thermocouple psychrometer.

     

Which water potential? Differences between isopiestic thermocouple psychrometer measurements of intact and excised plant materials

Water potentials of leaves from well-watered plants were measured. There were species-specific differences in both the total and the osmotic potentials of pea (Pisum sativum), tradescantia (Tradescantia versicolor), rose (Rosa hybrida), bitter lemon (Citrus aurantium) and olive (Olea europaea). With tradescantia the potential measured after the destruction of turgor by freezing was less negative than before, a result which suggests that the value obtained is not identical with the real osmotic potential of the leaf. detached leaves of all species showed less negative water potential readings, and those of pea even a less negative osmotic potential, when cut into five pieces than when measured intact. Application of vaseline to the cut surface of the leaves reduced this effect with rose and olive, though not with tradescantia and pea. Measurements were also made of the water potentials of comparable leaves of tradescantia and bitter lemon, attached to and detached from their plants; when bitter lemon leaves were detached and watered through their petioles which protruded outside the thermocouple chamber, their potential became considerably less negative than when the same leaves had been attached to well watered plants. However, similar leaves whose cut petioles were introduced into the thermocouple chamber registered an even less negative potential. The results are consistent with the hypothesis that when a leaf is cut off a plant, and even more so when it is cut into sections, the water previously held by matrix forces becomes available to dilute the “spilled” cell sap and to be absorbed by adjacent cells and thereby to increase their turgor and render the net water potential of the leaf less negative. Similarly, the apparent negative turgor of the succulent, tradescantia leaves is likely to be due to dilution of the osmotic component by cell wall water. The discrepancies between the readings of attached and detached leaves indicate a considerable whole-plant matrix component, and the results as a whole suglest that thermocouple psychrometer readings carried out on detached and even more on cut-up leaves may be artifacts and that it is desirable to determine water potentials on leaves attached to their plants. The work was supported by a Government of Israel Fellowship and was conducted at the Department of Pomology and Viticulture, Faculty of Agriculture of the Hebrew University of Jerusalem, Rehovot, Israel.     

Toxicity of Leaf and Stem Extracts to Tylenchorhynchus dubius

Plant extracts, made by grinding 2 g of fresh tissue in 5 ml of water, were toxic to Tylenchorhynchus dubius and Hoplolaimus spp. Such extracts from leaves and stems of bean (Phaseolus vulgaris L.) and leaves of tobacco (Nicotiana tabacum L.) were most toxic; those from leaves of corn (Zea mays L.), tomato (Lycopersicon esculentum Mill.) and rhododendron (Rhododendron catawbiense L.) were less toxic; and extracts of bean roots were nontoxic. Nematode movement slowed markedly within 1 hr in tobacco leaf extract, and within 4 hr in bean leaf extract; both extracts completely inactivated or killed 95% of the nematodes in 24 hr. Heating leaf extract 10 min at 80 C eliminated toxicity. Absorption of fusicoccin, a phytotoxin produced by Fusicoccum amygdali Del., increased the toxicity of tomato leaf extracts, whereas water extracts of acetone-extracted powder preparations of leaves were about 15-fold more toxic than water extracts of fresh tissue. Addition of homogenized leaves of bean, tobacco and tomato to soil significantly reduced nematode populations within 3 days.     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

Response of microbial activity and community structure to decreasing soil osmotic and matric potential

Low soil water content (low matric potential) and salinity (low osmotic potential) occur frequently in soils, particularly in arid and semi-arid regions. Although the effect of low matric or low osmotic potential on soil microorganisms have been studied before, this is the first report which compares the effect of the two stresses on microbial activity and community structure. A sand and a sandy loam, differing in pore size distribution, nutrient content and microbial biomass and community structure, were used. For the osmotic stress experiment, salt (NaCl) was added to achieve osmotic potentials from ?0.99 to ?13.13 MPa (sand) and from ?0.21 to 3.41 MPa (sandy loam) after which the soils were pre-incubated at optimal water content for 10d. For the matric stress experiment, soils were also pre-incubated at optimal water content for 10d, after which the water content was adjusted to give matric potentials from ?0.03 and ?1.68 MPa (sand) and from ?0.10 to 1.46 MPa (sandy loam). After amendment with 2% (w/w) pea straw (C/N 26), soil respiration was measured over 14d. Osmotic potential decreased with decreasing soil water content, particularly in the sand. Soil respiration decreased with decreasing water potential (osmotic?+?matric). At a given water potential, respiration decreased to a greater extent in the matric stress experiment than in the osmotic stress experiment. Decreasing osmotic and matric potential reduced microbial biomass (sum of phospholipid fatty acids measured after 14 days) and changed microbial community structure: fungi were less tolerant to decreasing osmotic potential than bacteria, but more tolerant to decreasing water content. It is concluded that low matric potential may be more detrimental than a corresponding low osmotic potential at optimal soil water content. This is likely to be a consequence of the restricted diffusion of substrates and thus a reduced ability of the microbes to synthesise osmolytes to help maintain cell water content. The study also highlighted that it needs to be considered that decreasing soil water content concentrates the salts, hence microorganisms in dry soils are exposed to two stressors.     

Occurrence of Inverted Water Potential Gradients between Soil and Bean Roots

Measurements with a pressure chamber were made of the xylem water potential of leaves, shoots and roots from bean plants (Pkaseolus vulgaris L. cv. Processor) grown with a 12 hour dark period and natural or artificial light conditions during the day. The water potentials were measured at the end of a dark period and during the light period. Measurements taken at the end of the dark period indicated normal potential gradients within the soil/plant system (leaf < shoot < root < soil), when the matric potential of soil water was relatively high (above ?0.02 bar), and the gradients then also remained normal during the day (natural light). When the soil water potential was ?1 bar or lower in the morning, however, the root xylem water potential was higher than the soil water potential; at very low soil water potentials (< ?4 bar) it remained higher during most of the day. In this case also leaf and shoot xylem water potentials were higher than the soil water potential in the early morning, although decreasing rapidly in daylight. Under artificial light, both leaf and root water potentials were higher than the soil water potential throughout the whole diurnal cycle when the latter potential was below ?4 bar. From measurements of stomatal diffusion resistance, transpiration, relative water content of leaves and of changes in the matric potential of soil water, it was concluded that when the matric potential of soil water was low, water could be taken up by the plant against a water potential gradient. Because leaf xylem water potential was always lower than root xylem water potential, the mechanism involved in the inversion of water potential gradient must be localized in the roots, and probably related to ion uptake. Symbols and abbreviations used in the text: Ψ: Plant water potential (thermocouple psychrometer); Ψx: Xylem water potential (pressure chamber); Ψs: Osmotic potential of xylem sap; Ψm: Matric potential of soil water; RWC: Relative water content.     

Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H₂O₂ and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.     

The gradient between pre-dawn rhizoplane and bulk soil matric potentials, and its relation to the pre-dawn root and leaf water potentials of four species

Uptake of soil water by plants may result in significant gradients between bulk soil and soil in the vicinity of roots. Few experimental studies of water potential gradients in close proximity to roots, and no studies on the relationship of water potential gradients to the root and leaf water potentials, have been conducted. The occurrence and importance of pre-dawn gradients in the soil and their relation to the pre-dawn root and leaf water potentials were investigated with seedlings of four species. Pre-germinated seeds were grown without watering for 7 and lid in a silt loam soil with initial soil matric potentials of -0.02, -0.1 and -0.22 MPa. Significant gradients, independent of the species, were observed only at pre-dawn soil matric potentials lower than -0.25 MPa; the initial soil matric potentials were -0.1 MPa. At an initial bulk soil matric potential of -0.22 MPa, a steep gradient between bulk and rhizoplane soil was observed after 7 d for maize (Zea mays L. cv. Issa) and sunflower (Helianthus annuus L. cv. Nanus), in contrast to barley (Hordeum vulgare L. cv. Athos) and wheat (Triticum aestivum L. cv. Kolibri). Pre-dawn root water potentials were usually about the same as the bulk soil matric potential and were higher than the rhizoplane soil matric potential. Pre-dawn root and leaf water potentials tended to be much higher than rhizoplane soil matric potentials when the latter were lower than -0.5 MPa. It is concluded that plants tend to become equilibrated overnight with the wetter bulk soil or with wetter zones in the bulk soil. Plants can thus circumvent negative effects of localized steep pre-dawn soil matric potential gradients. This may be of considerable importance for water uptake and growth in drying soil.     

The isolation, culture and regeneration of Petunia leaf protoplasts

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.     

Chloroplast response to low leaf water potentials: I. Role of turgor

The effect of decreases in turgor on chloroplast activity was studied by measuring the photochemical activity of intact sunflower (Helianthus annuus L. cv. Russian Mammoth) leaves having low water potentials. Leaf turgor, calculated from leaf water potential and osmotic potential, was found to be affected by the dilution of cell contents by water in the cell walls, when osmotic potentials were measured with a thermocouple psychrometer. After the correction of measurements of leaf osmotic potential, both the thermocouple psychrometer and a pressure chamber indicated that turgor became zero in sunflower leaves at leaf water potentials of −10 bars. Since most of the loss in photochemical activity occurred at water potentials below −10 bars, it was concluded that turgor had little effect on the photochemical activity of the leaves.     

In vitro water stress bioassays with the nematophagous fungus Pochonia chlamydosporia: Effects on growth and parasitism

The biocontrol potential of Pochonia chlamydosporia, a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P. chlamydosporia isolates. The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P. chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.     

Survey of the matric water of various plant groups

Matric water (Matric-bound water) was measured as the water retained by plant material after equilibration on a pressure membrane or ultra filter. At increasing pressures, lower amounts of water are held by matric or colloidal surface forces. Matric water was expressed as a percent of either (a) the dry weight or (b) the original water content. Plant material was oven dried, ground, and then saturated with water prior to the matric determination, which occurred in the pressure membrane filter at either one bar or 20 bars, supplied by cylinder nitrogen for 72 hours.Matric water was measured in leaves or in photosynthetic stem tissues from sixty species, including hydrophytes, xerophytes, and halophytes. The correlations between matric potentials and drought and salt tolerance ratings were determined. High matric-bound water values as well as high tissue water contents were found in species of Chenopodiaceae, which are considered more salt and drought tolerant than species of other plant groups. Low matric water values were found in the coniferous tree leaves and in grasses, and intermediate values were found in Compositae, miscellaneous herbaceous species, and deciduous trees. No correlation could be found between either salt or drought tolerance ratings and the matric percent when the latter was expressed on a dry weight basis. When matric water was expressed as a percent of the original water content, there was a positive correlation with drought tolerance ratings in all families studied except succulent Chenopodiaceae.Utah Agricultural Experiment Station Journal Paper No. 1229.     

An interpretation of some whole plant water transport phenomena

A treatment of water flow into and through plants to the evaporating surface of the leaves is presented. The model is driven by evaporation from the cell wall matrix of the leaves. The adsorptive and pressure components of the cell wall matric potential are analyzed and the continuity between the pressure component and the liquid tension in the xylem established. Continuity of these potential components allows linking of a root transport function, driven by the tension in the xylem, to the leaf water potential. The root component of the overall model allows for the solvent-solute interactions characteristic of a membrane-bound system and discussion of the interactions of environmental variables such as root temperature and soil water potentials. A partition function is developed from data in the literature which describes how water absorbed by the plant might be divided between transpiration and leaf growth over a range of leaf water potentials.

Relationships between the overall system conductance and the conductance coefficients of the various plant parts (roots, xylem, leaf matrix) are established and the influence of each of these discussed.

The whole plant flow model coupled to the partition function is used to simulate several possible relationships between leaf water potential and transpiration rate. The effects of changing some of the partition function coefficients, as well as the root medium water potential on these simulations is illustrated.

In addition to the general usefulness of the model and its ability to describe a wide range of situations, we conclude that the relationships used, dealing with bulk fluid flow, diffusion, and solute transport, are adequate to describe the system and that analogically based theoretical systems, such as the Ohm's law analogy, probably ought to be abandoned for this purpose.

     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

CO2 is the main inorganic C species entering photosynthetically active leaf protoplasts of the freshwater macrophyte Ranunculus penicillatus ssp. pseudofluitans

Submerged aquatic macrophytes growing in water where free CO₂ is unavailable (above pH 8·2) must use mechanisms to supply external dissolved inorganic carbon in a form available to chloroplasts (CO₂). Active transport of HCO₃^– across the plasmalemma has not been proven to be widespread in aquatic macrophytes and catalytic conversion of HCO₃^– to CO₂ is the usual supply mechanism in submerged macrophytes. The interaction of leaf form and function in this respect was investigated in the linear, submerged leaves of Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S.Webster. Viable protoplasts were isolated using a mixture of cell wall degrading enzymes optimized for this species. Protoplast viabilities greater than 80% after 5 h of isolation were achieved. Photosynthetic rates of isolated protoplasts were comparable with that of intact plant tissue. Results of carbon isotopic disequilibrium experiments showed that CO₂ was the preferred species of dissolved inorganic carbon for photosynthesis by protoplasts and that HCO₃^– which predominates in the plant’s natural environment mainly contributes by supplying CO₂ outside the cells.     

Effects of high-temperature stress on various biomembranes of leaf cells in situ and in vitro

The sensitivity of photosynthetic and respiratory functions to supraoptimal temperature stress was compared after heating of leaves, protoplasts and membrane systems of spinach (Spinacia oleracea L. cv. Monatol) and lettuce (Valerianella locusta [L.] Betcke) in situ and in vitro.

After heating of whole leaves or protoplasts, endogenous respiration was not or only slightly affected at temperatures which caused a marked decrease of photosynthesis. This was manifested when mitochondria and thylakoids were isolated from heat-treated leaves. In the presence of exogenous substrates, mitochondrial electron transport and phosphorylation were even somewhat stimulated compared to the controls.

Inactivation of net CO₂ uptake of whole leaves following heat stress and of the photochemical activities of chloroplast membranes isolated from heat-treated leaves of the same origin occurred nearly simultaneously. In protoplasts, photosynthesis was inactivated at temperatures far below those which caused drastic changes in the integrity of the tonoplast and the plasmalemma. This indicates that damage occurring within the chloroplasts rather than alterations in the compartmentation of the cell is responsible for the high sensitivity of photosynthesis to supraoptimal temperature stress.

Mitochondria and thykaloids isolated from the same preparation of intact leaves under comparable conditions and subjected to heat treatment in vitro, however, were inactivated nearly in the same temperature range. Thus, mitochondria are much more stable within their cytoplasmic environment.

     

Comparison of physiological responses of guard cell protoplasts of Nicotiana glauca isolated from leaves collected before dawn or at midday

We observed that guard cell protoplasts isolated from leaves collected at midday from Nicotiana glauca Graham (tree tobacco) did not give the same physiological responses to light as those isolated from leaves collected in early morning. Based on that observation, we attempted to determine whether there were significant differences between the physiological responses of guard cell protoplasts isolated from leaves collected before dawn (with closed stomata) and those isolated from leaves collected at midday (with open stomata). We isolated guard cell protoplasts from leaves collected before dawn and at midday and compared (1) rates of red and blue light-induced pH changes in weakly buffered media caused by changes in their metabolism, (2) their rates of oxygen consumption in darkness and oxygen evolution in light and (3) relative rates of decay of variable chlorophyll a fluorescence in their chloroplasts. Studies with the vital stain fluorescein diacetate failed to reveal any significant differences in the viabilities of protoplast preparations from leaves collected before dawn and at midday. Furthermore, protoplasts from leaves collected at these times swelled to similar extents in an osmotic medium containing 10 µM fusicoccin and 5 mM KCI. Nevertheless, rates of light-induced pH changes, rates of oxygen consumption and evolution and rates of decay of variable chlorophyll a fluorescence were all lower in preparations of guard cell protoplasts from leaves collected at midday than in preparations from leaves collected before dawn. Initial volumes of guard cell protoplasts isolated from leaves collected at midday were 150% of those of guard cell protoplasts isolated from leaves collected before dawn. We suggest that the differences in responses of guard cell protoplasts isolated from leaves collected before dawn and at midday may be caused by (1) nonoptimal isolation conditions for guard cell protoplasts prepared from leaves collected at midday, (2) the lower surface-to-volume ratio of guard cell protoplasts isolated from leaves collected at midday or (3) diurnal and/or circadian regulation of guard cell metabolism over the course of a day.