Showing papers in "Planta in 1975"

Nutritional Requirements for Growth of Vicia hajastana Cells and Protoplasts at a Very Low Population Density in Liquid Media

Abstract: When Vicia hajastana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/ml), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-dichlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-1-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 cells/ml in this defined medium. The population density could be lowered to 1-2 cells/ml with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 ml of this medium. Within 30-40 days, each one had grown indefinitely and formed a mass of cells (ca. 10(7)).

The Control of Glutamine Synthetase Level in Lemna minor L.

Abstract: The specific activity of glutamine synthetase (E.C. 6.3.1.2) of Lemna minor L. is markedly reduced when either ammonium ions or glutamine are present in the growth medium. Combinations of 5 mM ammonia and 5 mM glutamic acid or 5 mM ammonia and 5 mM glutamine as nitrogen source, lead to a 4–5 fold reduction of the maximum activity measurable on 5 mM γ-aminobutyric acid. Analyses of the soluble pool of nitrogen indicate that the reduction in enzyme level is associated with an increase in the pool of glutamine. There is an inverse correlation between the apparent rate of synthesis of glutamine synthetase and the intracellular concentration of glutamine, and this relationship suggests that the glutamine synthetase of Lemna minor is subject to end product repression by the endogenous pool of glutamine.

Simultaneous Requirement of Carbon Dioxide and Abscisic Acid for Stomatal Closing in Xanthium strumarium L.

Abstract: Open stomata of detached leaves of Xanthium strumarium L. closed only when carbon dioxide and abscisic acid (ABA) were presented simultaneously. Three parameters of stomatal closing were determined after additions of ABA to the irrigation water of detached leaves, while the leaves were exposed to various CO2 concentrations ([CO2]s) in the air; a) the delay between addition of ABA and a reduction of stomatal conductance by 5%, b) the velocity of stomatal closing, and c) the new conductance. Changes in all three parameters showed that stomatal responses to ABA were enhanced by CO2; this effect followed saturation kinetics. Half saturation occurred at an estimated [CO2] in the stomatal pore of 200 μl l-1. With respect to ABA, stomata responded in normal air with half their maximal amplitude at [ABA]s between 10-6 and 10-5 M(+-)-ABA. The amounts of ABA taken up by the leaves during the delay increased with a power <1 (on the average, 0.67) of the [ABA] in the transpiration stream. The minimal amount of ABA found to produce a stomatal response was about 1 pmol of (+-)-ABA per cm2 leaf area, almost two orders of magnitude smaller than the original content of the leaves in ABA indicating that most of the endogenous ABA was in a compartment isolated from the guard cells. An interaction between stomatal responses to CO2 and ABA was also found in Gossypium hirsutum L. and Commelina communis L.; it was however much weaker than in X. strumarium. Based on earlier findings and on the results of this investigation it is suggested that stomata close if the cytoplasm of the guard cells contains much malate and H+. The acid content in turn is determined by the relative rates of production of malic acid (from endogenous as well as exogenous CO2) and its removal (by transport of the anion into the vacuole and exchange of the H+ for K+ with the environment of the guard cells). The simultaneous requirement of CO2 and ABA for stomatal closure leads to the inference that ABA inhibits the expulsion of H+ from guard cells.

Patterns of nitrogen utilization in the soybean.

Abstract: The patterns of nitrate uptake, nitrate reductase activity in the leaves, and nitrogen fixation by the nodules were investigated in field-grown soybeans (Glycine max (L.) Merr.) over the growing season. The level of nitrate-reductase activity generally paralleled the concentration of nitrate in the leaf tissue over the entire growing season. A precipitous drop in both parameters was noted within 2–3 weeks after flowering. These parameters decreased by 80–95% at mid-pod fill, a stage where ovule (seed) development was in the logarithmic growth phase, placing a heavy demand on the plant for both energy and fixed nitrogen. The activity of nitrogen fixation of soybean root nodules bore a reciprocal relationship to that of nitrate reductase. The maximum levels of nitrogen fixation were reached at early pod fill when nitrate reductase activity had dropped to 25% of maximum activity. A rapid loss of nitrogen fixation activity occurred shortly after bean fill was initiated, again at a time when the ovules were developing at maximal rates. The total protein content of soybean leaves increased over the season to a maximum level at mid-pod fill. This was followed by a 50% drop over the next 3-week period when the plants approached senescence. This drop corresponded to that found for nitrogen fixation. A similar pattern was noted for watersoluble proteins in the leaf. These studies suggest that there is a close and competitive relationship between the processes of nitrate reduction and nitrogen fixation, with the latter process dominating as the major source of fixed nitrogen after the plants have flowered and initiated pods. At this transitional stage, both soil and environmental effects could cause pertrubation in these processes that could lead to a nitrogen stress causing flower and pod abscission. The rapid decay of nitrogen fixation at the time of midpod fill also suggests a competition between roots (nodules) and pods for available photosynthate. This competition appears to lead to the breakdown of foliar proteins and senescence.

Seasonal changes in structure and function of spruce chloroplasts.

Abstract: Seasonal changes of ultrastructure were studied by electron microscopy and by determining the chlorophyll and starch content of the plastids. Young plastids of spruce (Picea abies (L.) Karst.) first function as amyloplasts which store reserve material for the growth of the young needles. Then they develop a normal thylakoid system and produce assimilation starch during the day. In autumn, starch synthesis ceases and the plastids group together. In winter they swell and their membrane system becomes disorganized and reduced. With increasing temperatures in spring the chloroplasts recover, but then they accumulate large amounts of starch, which is not broken down during the night or even during a dark period of several days. As in the previous year they now function as amyloplasts providing reserve material for the new shoot. In summer these plastids are again converted into typical chloroplasts. The same seasonal changes of structure and function could be observed in chloroplasts from 2- or 3-year old needles. Thus these changes represent cyclic processes, which repeat each year. Features of slow aging are superimposed on to these cycles.

Xylem to phloem transfer of solutes in fruiting shoots of legumes, studied by a phloem bleeding technique

Abstract: Comparisons were made of the levels of various solutes in xylem (tracheal) sap and fruit tip phloem sap of Lupinus albus (L.) and Spartium junceum (L.). Sucrose was present at high concentration (up to 220 mg ml-1) in phloem but was absent from xylem whereas nitrate was detected in xylem (up to 0.14 mg ml-1) but not in phloem. Total amino acids reached 0.5–2.5 mg ml-1 (in xylem) versus 16–40 mg ml-1 in phloem. Phloem: xylem concentration ratios for mineral nutrients (K, Na, Mg, Ca, Fe, Zn, Mn, Cu) spanned the range 0.7 to 20, the ratios generally reflecting an element's phloem mobility and its availability to the xylem from the roots.

Compositional changes in developing rape seed (Brassica napus L.).

Abstract: The growth and composition of siliquas and seeds of oilseed rape was followed over 12 weeks from shortly after anthesis to maturity. Each plant produced 220 siliquas, this number being constant throughout development. Seed numbers per siliqua fell from 19 to 9 by week 5 and declined to 7 at maturity. Hull1 and seed growth followed a sigmoid pattern, but were not in phase. Seed development could be divided into 3 phases: In Phase 1, seed weight was low and starch and ethanol soluble compounds accounted for 80% DM. Phase 2, seed growth increased and storage oil and proteins were deposited accounting for 40% and 20% DM respectively at the end of this stage. Starch, glucose and fructose were utilized in this process. Phase 3 was largely concerned with the deposition of oil and protein in fixed proportions. Seed weight more than doubled while DM composition remained constant. Sugars were transferred from the hull to the seed to support this growth.

Relative thermostability of the chloroplast envelope

Abstract: Intact isolated chloroplasts from leaves of Spinacia oleracea L. were subjected to heat treatment. After heating, the integrity of the chloroplast envelopes and the activities of various light-dependent chloroplast reactions were tested. The integrity of the chloroplast envelopes, as judged from rates of ferricyanide reduction, enzyme compartmentation and visual appearance of the chloroplasts in the light microscope with phase optics, was affected much less by heat stress than the photochemical reactions of thylakoids. This indicates a comparatively high thermostability of the chloroplast envelope membranes. It is also evidence of a differential thermostability of different biomembranes. Photophosphorylation was highly susceptible to thermal stress. Heat treatment that partly inactivated phosphorylation stimulated light-dependent quenching of 9-aminoacridine fluorescence, which served as an indicator of proton transfer from stroma to thylakoids in intact chloroplasts. Drastic changes in the characteristics of chlorophyll a fluorescence emission caused by heating were probably due to structural alterations of the thylakoid system.

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L.

Abstract: From compartmental analysis of radioisotope elution measurements, concentrations and fluxes of K+, Na+ and Cl- were estimated for cortical cells in root segments of onion, Allium cepa L., relative to a complete nutrient solution. The transported fraction of the total efflux was estimated separately. With the Ussing-Teorell flux ratio equation as the criterion, it was concluded that all three ions were actively accumulated from the outside medium into the cytoplasm and that only Na+ was actively accumulated into the vacuole. K+ and Cl- moved passively, in both directions across the tonoplast. Failure to account for leakage from the stele via the segment cut ends resulted in an over-estimate of exchange across the tonoplast but did not alter the conclusions qualitatively. The consequences of changing the assumed value of the tonoplast electrical potential (from 0 to+10- mV), and the effects of different experimental procedures, were also assessed, and found not to affect the main conclusions significantly. Separate measurement of ions leaking from the segment ends revealed that Na+ was transported almost exclusively in an acropetal direction in the stele. Cl- appeared at both ends of the segments in similar amounts and K+ was transported mainly in the basipetal direction. The implications of these findings for the mechanism and site of ion selectivity are discussed.

Histochemistry and fine structure of developing wheat aleurone cells.

Abstract: Developing aleurone cells can first be distinguished 10 days after anthesis beneath the degenerating nucellus as somewhat cuboidal cells with extremely thin walls and large nuclei. Ribosomes are very abundant but little endoplasmic reticulum (ER) is apparent. By 14 days the cell walls are intensely autofluorescent, possibly due to the presence of a ferulic acid-carbohydrate complex. At this stage the cytoplasm is characterized by the presence of large vacuoles, many of which contain small, electron-dense inclusions, presumably the beginnings of the phytin globoids (Type I inclusions) of mature aleurone grains. The paired appearance of many of the cells suggests that they are dividing periclinally, the innermost cells destined to become part of the starchy endosperm. By 4 weeks the cell walls have greatly thickened, ER and mitochondria have proliferated, and the vacuoles, which subsequently give rise to mature aleurone grains, contain a second type of inclusion (Type II inclusion) embedded in a protein matrix. Although the walls remain uniformly autofluorescent, an intensely stained inner wall can be distinguished readily from the outer wall. By 5 weeks the aleurone grains are almost completely surrounded by lipid droplets and contain numerous Type I inclusions. The cells change little in appearance from 6 weeks to maturity. At the latter stage the inner and outer walls are quite distinct and the cytoplasm is densely packed with aleurone grains which are completely surrounded by lipid droplets and interspersed with occasional plastids and numerous mitochondria with rather indistinct cristae.

Correlative Inhibition of Lateral Bud Growth in Phaseolus vulgaris L. Timing of Bud Growth Following Decapitation

Abstract: On intact, 3-week-old plants of Phaseolus the larger bud in the axils of the primary leaves shows slow, continuous elongation growth. Release from correlative inhibition can be detected within 30 min following decapitation. When 0.1% indoleacetic acid in lanolin is applied to the decapitated stem stump, the lateral bud shows slow growth during the first 7 h, then stops completely for a further 15 h but after 2 days a further gradual increase in length is observed.

The development of proteolytic activity and protein degradation during the germination of Pisum sativum L.

Abstract: The change in protein content and composition of the cotyledons of Pisum sativum L. cv. Burpeeana during germination was studied. Protein depletion from the cotyledons was slow during the first 4 days of germination but became rapid on the 5th day and by the 16th day the majority of the protein had disappeared. During the first 4 days the depletion of the globulins exceeded that of the albumins; legumin appeared to be degraded slightly more rapidly than vicilin during the early phase of germination. Sodium-dodecylsulfate (SDS) electrophoresis of SDS- and dithiothreitol-dissociated globulins indicated that before rapid protein depletion there were marked changes in the component composition of the major globulins legumin and vicilin. The onset of rapid protein depletion was associated with an increase in the level of an acid-sulfhydryl protease in the cotyledons. These findings indicate that the reserve globulins undergo modifications prior to their eventual hydrolysis.

The pattern of development of Anabaena in the Azolla-Anabaena symbiosis

Abstract: The development of Anabaena in the leaf cavities of Azolla (probably A. filiculoides) was studied. After the alga is installed in the cavity, heterocyst frequency rises to a maximum (20–30%) about 12 leaves from the apex, then remains constant until the leaf senesces. The size of vegetative cells of the alga increases with cell width apparently increasing linearly with increasing leaf age.

Abscisic acid as a root growth inhibitor: Physiological analyses.

Abstract: Abscisic acid (ABA) moves basipetally and laterally in maize (Zea mays L.) root segments placed horizontally; its transport properties are thus similar to those of the growth-inhibiting substances produced by the root cap. The two opposite flows af ABA and of indolyl-3-acetic acid (IAA) — substances both present in the cap — may control elongation and georeaction of the root.

Temperature effects on the carbon-isotope ratio of C3, C 4 and crassulacean-acid-metabolism (CAM) plants.

Abstract: The temperature coefficient of the δ13C value over the temperature range 14 to 40° has been measured for plants with different photosynthetic pathways. C3 plants (6 species) had a temperature coefficient of-0.0125‰/o, C3 plants (5 species)-0.046‰/o, “C4-like” CAM (2 species)-0.13‰/o and “C3-like” CAM (2 species)-0.019‰/o. The relative temperature insensitivity of the δ13C value is in contrast with the measured temperature dependence of δ13C values for ribulose-diphosphate carboxylase in vitro, and to the temperature response of photosynthesis. The temperature sensitivity of the “C4-like” CAM plants was the relatively greatest; this is consistent with the evidence that high night temperature inhibits dark CO2 fixation. The same inhibition may occur in the “C3-like” CAM plants but it would not have as significant an effect on the δ13C value as these plants are primarily dependent on primary fixation of exogenous CO2 via the ribulose-diphosphate carboxylase reaction.

Organogenesis from Callus Culture of Hordeum vulgare

Abstract: Rapidly proliferating callus cultures from the apical meristem of Hordeum vulgare L., cultivars “Himalaya” and “Mari”, were established on a defined medium supplemented with auxin and cytokinin. A frequency of organ differentiation of ca. 85% was obtained by transfer to the same medium but without any growth regulators. All regenerated plants had the diploid chromosome number of 14 (2n).

Growth regulator levels in embryo and suspensor of Phaseolus coccineus at two stages of development.

Abstract: Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5-6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4-0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3-0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.

Developmental studies on microbodies in wheat leaves : III. On the photocontrol of microbody development.

Abstract: 1. In etiolated wheat (Triticum aestivum L.) leaves, the development of the microbody enzymes catalase, hydroxypyruvate reductase, and glycolate oxidase was specifically stimulated by short treatments of the seedlings with red light, although the increases were less than observed after treatment with continuous white light. A comparison of the effects of short red and far-red exposures indicated the involvement of phytochrome. 2. Continuous far-red light treatments also enhanced the development of microbody enzymes. Catalase activity continued to increase at a high rate even after return from a prolonged far-red illumination to darkness, while the increase in the activities of glycolate oxidase and hydroxypyruvate reductase fell to the dark rates when the tissue was removed from the light. However, even at higher intensities of continuous far-red light the microbody enzymes reached only considerably lower activities than in white light. During continuous irradiation of equal quantum flux, the microbody enzymes reached higher activities in red than in far-red light, but the highest activities were observed in blue light, which had similar effects as white light. The quantitative difference between the effects of prolonged red or blue light depended also on the seed material and growing conditions. In the presence of the herbicide 3-amino-1,2,4-triazole the increase of glycolate-oxidase activity was reduced in red light but was affected much less, if at all, in blue light. 3. Continuous irradiations with all three light qualities used (red, far-red, blue) influenced the properties of the microbody particles to form a distinct band sharply confined close to an equilibrium density of 1.25 g cm-3 on sucrose gradients which was not observed in preparations from plant material raised in complete darkness. In preparations from all light-grown plants a special peak in the activity profile of malate dehydrogenase was found in the microbody fraction while it was lacking on gradients from dark-grown leaves. The heights of the activities of malate dehydrogenase as well as of the other enzymes found in the microbody fractions from plants grown in either far-red, red, or blue light differed in the same way as did the activities from total leaf homogenates. 4. Glycolate oxidation by segments of intact leaf tissue was higher with tissue from light- than from dark-grown plants, but after light treatments of different spectral quality its magnitude did not correspond to the extractable activities of glycolate oxidase.

The use of compartmental analysis in the study of the movement of carbon through leaves.

Abstract: The export of 14C from leaves of Lycopersion esculentum (Mill.), Capsicum frutescens (L.) and Amaranthus caudatus (L.) was followed by in vivo counting after exposure of the leaf to a 5 min pulse of 14CO2. In all instances the time course of export showed two or more exponential phases. There was an initial rapid period of export which was followed by a slower phase after about 2 h. About 12–14 h after exposure to 14CO2 this second phase was superseded by an even slower phase of export which continued for more than 24 h. In tomatoes the initial phase was most rapid in plants bearing fruit which had been heated to 30°C instead of the standard 15–20°C; it was slowest when the fruit were removed. In Amaranthus the rate of the initial phase was shown to be positively correlated with photosynthesis and when the latter was prevented by either darkness or the absence of CO2 the rate of loss of 14C was reduced. The data were used to test a model of carbon movement from a leaf which postulated the presence of two carbon pools which turned-over at different rates. The photosynthetic carbon entered the pool with the faster rate of turn-over—the ‘labile’ pool—and exchanged with the other, ‘storage’, pool. Export from the leaf was from the ‘labile’ pool. The results suggested that a third, longer term, storage pool should be included in the model and that the exchange between the pools should be non-linear.

Auxin-induced hydrogen ion excretion: correlation with growth, and control by external pH and water stress.

Abstract: 1. The acid-growth theory predicts that the rates of auxin-induced cell elongation and H+-excretion should be closely correlated as long as the experimental conditions remain fairly constant. To test this, Avena coleoptiles have been induced to elongate at different rates by varying the concentration of auxin, the age of the tissue, or by addition of metabolic inhibitors. As predicted, in each case there was a close correlation between the rates of H+-excretion and growth. 2. The rate and direction of movement of H+ between the coleoptile and the external medium is regulated by the external pH. Coleoptiles take up H+ passively from acidic solutions and excrete H+ into basic solutions. In the absence of auxin, uptake and excretion are at equilibrium when the solution pH is near 5.7, a pH too high to allow rapid cell wall loosening. Auxin stimulates the excretion, but as the external pH drops the excretion is inhibited and a new equilibrium is established near 5.0. This allows amlows maximum wall loosening without causing toxic side-effects. 3. H+-excretion is also affected by water stress. Increasing water stress induced by mannitol decreases H+-excretion in auxin-treated tissues but stimulates H+-excretion in the absence of auxin. At incipient plasmolysis H+-excretion is insensitive to auxin, suggesting that even if H+-excretion is mediated by an auxin-activated ATPase it may be impossible to demonstrate an effect of auxin on this enzyme in vitro. 4. Three types of H+-excretion have been recognized in coleoptiles; auxin-induced, stress-induced, and basal level. All appear to require ATP, but only basal level H+-excretion does not also require continual protein synthesis.

Localisation of peroxidase isoenzymes in protoplasts and cell walls of Nicotiana tabacum L.

Abstract: Upon disk-electrophoresis with guaiacol as a substrate the peroxidase-isoenzymes of Nicotiana tabacum (L.) were localized on the gels in two anodic and two cathodic groups. By preparation of protoplasts and isolation of cell walls it was possible to show that only cathodic enzymes are located in the protoplasts in measurable amounts, whereas all the isoenzymes, anodic and cathodic, can be found associated with cell walls. The different groups of isoenzymes are bound to the cell wall in different ways as evidenced by differences in their extration. It seems possible that different biological functions are associated with the different groups of isoenzymes.The isoenzyme patterns of different organs and tissues of tobacco show qualitative differences only in the anodic (i.e. wall located) isoenzymes. It is suggested that the ontogenetic change in peroxidase-patterns is direct evidence of biochemical differences in the cell walls of the different tissues and organs.

Effect of osmotic stress on abscisic acid levels in xylem sap of sunflower (Helianthus annuus L.).

Abstract: Addition of an osmoticum (-12 bars) to the rooting medium of sunflowers (Helianthus annuus L.) caused an increase in the level of abscisic acid (ABA) present in xylem exudate subsequently collected from cut shoots. Using tall and dwarf plants it was shown that there was a time lag in the appearance of increased levels of ABA in tall plants when compared with dwarf plants. The results indicate that the leaves, rather than the roots are the site of synthesis of ABA present in the xylem sap of osmotically stressed sunflower plants.

Further studies on the relationship between the rates of nitrate uptake, growth and conductivity changes in the medium of plant cell suspension cultures.

Abstract: The changes in packed cell volume and in nitrate content and conductivity of the medium during the growth cycle of cell suspension cultures from Petroselinum hortense Hoffm., Glycine max Merr., and Haplopappus gracilis A. Grey in a chemically defined medium were compared. In all three cases sigmoidal curves obtained for large decreases in the conductivity were paralleled by similar curves for the rates of nitrate depletion from the medium until this nutrient was completely exhausted. Further decreases in the conductivity subsequent to nitrogen starvation proceeded at relatively slow rates and ceased when the cultures entered into the stationary phase of the growth cycle. Thus the previously reported method of deriving growth curves indirectly from conductivity measurements (Hahlbrock and Kuhlen, Planta 108: 271-278, 1972; Hahlbrock et al., Planta 118: 75-84, 1974) might be generally applicable for this particular medium. The method seems to be based on a continuous uptake by the cells of ionic constituents throughout all stages of actual growth, even beyond the stage of nitrate exhaustion.Cell suspension cultures from Cicer arietinum L. and Acer pseudoplatanus L. in two different, more complex media were used for similar experiments, in which the changes in packed cell volume and in the conductivity of the medium were recorded. As with the results obtained with the fully synthetic medium, the mirror-images of the curves obtained for the decline in conductivity initially paralleled the growth curves. However, the two curves became incongruous after a certain growth stage was reached. These results are discussed with respect to the composition of the media used and to the apparent limitations of the method of determining specific growth stages by monitoring conductivity changes in the medium.

Photosynthesis and photorespiratory CO2 evolution of water-stressed sunflower leaves.

Abstract: Rates of true photosynthesis (TPS), apparent photosynthesis (APS) and photorespiration (PR) of sunflower (Helianthus annuus L., Var. Mennonite) leaves were measured in air (21% O2, 300 vpm CO2) at 25° C and 400 μEinsteins m-2 s-1 radiant flux density. The plants were water stressed by application of osmoticum (polyethylene glycol 4000) to the root system. TPS and APS decreased linearly from maxima at-4 bar leaf-water potential (ψ) to become very small and zero respectively at about-18 bar ψ; at smaller potential CO2 was evolved from the leaf. Statistical analysis shows that TPS and APS were more closely correlated with ψ than stomatal conductance (r s -1), because r s -1 changed only in the range-4 to-13 bar but ψ exerted an effect at smaller potential. Photorespiration decreased linearly with stress and at-18 bar was 30% of the control plant rate; ψ and TPS accounted for only part of the variance in PR, both independently and in combination, and r s -1 accounted for little of the variance. Tricarboxylic acid cycle respiration of leaves placed for 20 min in darkness, remained almost constant with changing ψ and r s -1. It was one-third of photorespiration in control plants but increased as a proportion in severely stressed plants. The relative specific activity (RSA) of the CO2 released by PR of wellwatered plants was 90% after 20 min photosynthesis in 14CO2 but decreased to 18% at-18 bar ψ. Therefore, under stress mpre CO2 was derived by respiration from reserve materials and less from immediate photosynthate. Elimination of CO2 production by the glycollate pathway with small oxygen concentration (1.5%), showed that the contribution of TCA cycle respiration to photorespiration was small in unstressed plants but increased at small ψ to almost the same rate as photorespiration. It is concluded that desiccation decreased photosynthesis by decreasing the stomatal conductance to CO2 diffusion and by changing the balance between CO2 assimilation and production of the leaf. As a consequence carbon flux through the glycollate pathway decreased as did the rate of CO2 produced by it. However, TCA cycle respiration in the light increased with stress, so that total photorespiration remained large. The importance of maintaining carbon flux through the glycollate pathway and TCA cycle is discussed.

Leaf development and phloem transport in Cucurbita pepo: Carbon economy

Abstract: Net photosynthesis, dark respiration and growth for leaf 5 of Cucurbita pepo L. plants grown under controlled conditions were measured and the data used for an assessment of the changes in carbon balance during growth of the leaf through expansion to maturity. The blade is first capable of net CO2 fixation when ca. 8% expanded but the initial rapid growth during this period is sustained almost entirely through imported nutrients. When the growth rate starts to decline rapidly the net photosynthetic capacity of the blade begins to increase. This increase is accompanied by an expansion of the intercellular spaces and by decreasing dark respiration measured at night and in dark periods during the day. The blade becomes completely independent of phloem imported nutrients and begins to export excess photosynthate when the phase of rapid decrease in relative growth rate is almost complete at about 45% expansion. Maximum net photosynthesis of ca. 11 mg CO2 h(-1) dm(-2) is achieved at 70% expansion. The first detectable synthesis of the transport sugars stachyose and raffinose in the blade coincides with the beginning of intralaminar phloem transport from the tip to the base of the leaf. The synthesis of sucrose, the other major transport sugar, is detectable at all stages of leaf development.

Cortical cell fluxes and transport to the stele in excised root segments of Allium cepa L. : II. Calcium

Abstract: From compartmental analysis of radioisotope elution measurements, concentrations and fluxes of Ca(2+) were estimated for cortical cells in root segments of onion, Allium cepa L., relative to a complete nutrient solution containing 1 mM Ca(2+). Five compartments for Ca(2+) in the cortex were revealed. These were identified, in order of increasing rates of exchange, with the vacuole and cytoplasm of the cortical parenchyma, the Donnan free space in the cell walls, the water free space in the tissue and the superficial film of solution on the segments. With the Ussing-Teorell flux ratio equation as the criterion, it was concluded that Ca(2+) entered the cytoplasm passively and was actively pumped back to the external solution. Ca(2+) concentration in the vacuole could only be estimated as lying between wide limits (1.0 to 7.5 μeq. ml(-1)), but even at the maximum concentration, it was concluded that entry was passive and content limited by an efflux pump across the tonoplast. Net flux was zero and the vacuolar concentration of Ca(2+) compatible with this was found to be 2.6 μeq. ml(-1). The transported fraction of the total efflux, appearing at the segment cut ends, was estimated separately. Calcium was found to be transported almost exclusively in the basipetal direction.

Water relations of the epidermal bladder cells of the halophytic species Mesembryanthemum crystallinum: Direct measurements of hydrostatic pressure and hydraulic conductivity.

Abstract: Water exchange between the bladder cells of stems of the halophytic species Mesembryanthemum crystallinum and the subepidermal cortical cell layers was determined by direct cell turgor pressure measurements using a pressure probe. Treating the system bladder cell/subepidermal cortex as two homogeneous elastic compartments the hydraulic conductivity, L p, of the barrier separating both compartments was found to be L p=2·10-6 cm ·s-1·bar-1. As discussed, this value essentially reflects the hydraulic conductivity of the bladder cell membrane, which is unusually high compared with the values expected for higher plant cells. The L p-values did not show a dependence on cell turgor pressure over a large pressure range (0.1 to 6 bar) nor on the salinity of the bladder sap (osmolarity range: 500 to 1700 mOsmol). Furthermore, the stationary pressure-values obtained in the pressure-flow experiments point to unusually high permeabilities of the bladder membrane to electrolytes (KCl and NaCl). The unique transport properties of the bladder cell membrane for salt and water suggest a special physiological function of the bladders in the water economy of the plant. It is assumed that the bladders form a reservoir for salts and water in the plant which upon osmotic stress acts as a buffering system to protect the photosynthetic tissue from osmotic injury.

Formation of p-coumaric acid and o-coumaric acid from L-phenylalanine by microsomal membrane fractions from potato: Evidence of membrane-bound enzyme complexes.

Abstract: The enzymes described here are the membrane-bound L-phenylalanine ammonia-lyase and cinnamate hydroxylase. Microsomes prepared from tubers of Solanum tuberosum L. are capable of converting L-phenylalanine into both o- and p-coumaric acid. Three microsomal fractions obtained by density gradient centrifugation were characterized by their equilibrium densities. Within various subfractions we found different patterns of distribution of the two enzymes, probably because of the extent of their fixed arrangement on a membrane area. Simultaneous incubation of the microsomal fraction with L-[4-ring-(3)H]-phenylalanine and trans-[3-(14)C]cinnamate indicated the existence of two pools of substrate available to cinnamate p-hydroxylase: cinnamate formed by the L-phenylalanine-ammonialyase reaction was a more effective substrate than cinnamate added to the incubation mixture. We conclude that the coumaric acids are formed from L-phenylalanine by a mechanism by which endogenously formed cinnamate is only partially equilibrated with exogenous cinnamate supplied in the incubation medium. This effect of enzyme cooperation is dependent on the integrity of membranes. The extent of cooperation was reduced by attempts to purify the microsomal membranes and by treatment in vivo with ethylene at high concentrations.

Budding and cleavage division of tobacco mesophyll protoplasts in relation to pseudo-wall and wall formation.

Abstract: Two saline media, differing primarily in the presence or absence of NH4 + but also in the concentration of sucrose, were developed for culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts. In the R0.6 medium, which does not contain NH4 + and only 1 g/l sucrose, protoplasts divide 2–3 times by budding and form only a pseudo-wall, i.e. a nonrigid structure containing polysaccharides. Later the cells degenerate, and sustained division does not take place. In the W 0.6 medium, which contains NH4 + and 30 g/l sucrose, the protoplasts form a rigid wall and divide by cleavage of the cells. After a few divisions, the walls of practically all of the newly formed cells degenerate into pseudo-walls, and the divisions cease. Only a few cells keep a wall, continue to divide, and form colonies. A very high frequency of colony formations from protoplasts is obtained by culturing protoplasts for a week in R0.6 or W 0.6 and then diluting the culture with a sugar medium. A detailled study of the inorganic and organic components of the saline media showed a strong interaction between the nitrogen supply and the cytokinin requirement. The advantages of the saline media in obtaining cell colonies from protoplasts, the problems associated with budding-type division, the causes of the cessation of division when no complete wall is formed, and the conditions necessary for wall formation are discussed.

A Photoperiodic Response Mediated by Blue Light in the Brown Alga Scytosiphon lomentaria

Abstract: The crustose phase of Scytosiphon lomentaria (Lyngb.) J. Ag. persists indefinitely in 16 h of white light per day, but produces erect, cylindrical thalli vegetatively in 8-h days. The critical daylength for this short-day (SD) response is sharply defined, and, between 12 and 13 h, differences in daylength of only 15 min produce substantial differences in response. A significant response to SD can be induced by as few as 4 SD cycles, but 10–12 cycles are required to saturate the response and induce more than 90% of the plants to form thalli. The response to SD is completely inhibited by a 1-min light-break with a low irradiance of blue light, given in the middle of a 16-h dark period, but is unaffected by longer periods and higher irradiances of red or far-red light. There is good reciprocity between the irradiance and the length of a light-break with blue light, and 50% inhibition of the response to SD is induced by about 2 nE cm-2 at 449 nm. All attempts to reverse the inhibitory effects of blue light by subsequent irradiation with another wavelength have so far failed. These results indicate that phytochrome is not the photoreceptor pigment for this response, in spite of the similarity of the response in all other respects to the photoperiodic responses of flowering plants and other algae.