Showing papers in "Planta in 1985"

Establishment and maintenance of friable, embryogenic maize callus and the involvement of L-proline.

Abstract: Friable, embryogenic maize (Zea mays L.), inbred line A188, callus was established and maintained for more than one year without apparent loss of friability or embryogenic potential. Embryoid development was abundant in these cultures and plants were easily regenerated. Frequencies of friable-callus initiation and somatic-embryoid formation increased linearly with addition to N6 medium (C.C. Chu et al. 1975, Sci. Sin. [Peking] 18, 659–668) of up to 25 mM L-proline. Proline additions up to 9 mM to MS medium (inorganic elements of T. Murashige and F. Skoog 1962, Physiol. Plant. 15, 473–497, plus 0.5 mg 1-1 thiamine hydrochloride and 150 mg 1-1 L-asparagine monohydrate) did not stimulate embryoid formation. A major part of the difference between MS and N6 media could be attributed to their respective inorganic nitrogen components. L-Glutamine was not a satisfactory substitute for L-proline. Of 111 regenerated plants grown to maturity from three independent friable, embryogenic cell lines ranging in age from three to seven months, only four plants were abnormal based on morphology and pollen sterility. Seed was produced by 77% of the regenerated plants.

Effect of temperature on the CO2/O 2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase and the rate of respiration in the light : Estimates from gas-exchange measurements on spinach.

Abstract: Responses of the rate of net CO2 assimilation (A) to the intercellular partial pressure of CO2 (p i ) were measured on intact spinach (Spinacia oleracea L.) leaves at different irradiances. These responses were analysed to find the value of p i at which the rate of photosynthetic CO2 uptake equalled that of photorespiratory CO2 evolution. At this CO2 partial pressure (denoted Г), net rate of CO2 assimilation was negative, indicating that there was non-photorespiratory CO2 evolution in the light. Hence Г was lower than the CO2 compensation point, Γ. Estimates of Г were obtained at leaf temperatures from 15 to 30°C, and the CO2/O2 specificity of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (E.C. 4.1.1.39) was calculated from these data, taking into account changes in CO2 and O2 solubilities with temperature. The CO2/O2 specificity decreased with increasing temperature. Therefore we concluded that temperature effects on the ratio of photorespiration to photosynthesis were not solely the consequence of differential effects of temperature on the solubilities of CO2 and O2. Our estimates of the CO2/O2 specificity of RuBP carboxylase/oxygenase are compared with in-vitro measurements by other authors. The rate of nonphotorespiratory CO2 evolution in the light (R d ) was obtained from the value of A at Г. At this low CO2 partial pressure, R d was always less than the rate of CO2 evolution in darkness and appeared to decrease with increasing irradiance. The decline was most marked up to about 100 μmol quanta m-2 s-1 and less marked at higher irradiances. At one particular irradiance, however, R d as a proportion of the rate of CO2 evolution in darkness was similar in different leaves and this proportion was unaffected by leaf temperature or by [O2] (ambient and greater). After conditions of high [CO2] and high irradiance for several hours, the rate of CO2 evolution in darkness increased and R d also increased.

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes.

Abstract: In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K+ uptake by roots. When F1 hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.

Effects of salt stress on the growth, ion content, stomatal behaviour and photosynthetic capacity of a salt-sensitive species, Phaseolus vulgaris L.

Abstract: Phaseolus vulgaris (cv. Hawkesbury Wonder) was grown over a range of NaCl concentrations (0–150 mM), and the effects on growth, ion relations and photosynthetic performance were examined. Dry and fresh weight decreased with increasing external NaCl concentration while the root/shoot ratio increased. The Cl- concentration of leaf tissue increased linearly with increasing external NaCl concentration, as did K+ concentration, although to a lesser degree. Increases in leaf Na+ concentration occurred only at the higher external NaCl concentrations (≧100 mM). Increases in leaf Cl- were primarily balanced by increases in K+ and Na+. X-ray microanalysis of leaf cells from salinized plants showed that Cl- concentration was high in both the cell vacuole and chloroplast-cytoplasm (250–300 mM in both compartments for the most stressed plants), indicating a lack of effective intracellular ion compartmentation in this species. Salinity had little effect on the total nitrogen and ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) content per unit leaf area. Chlorophyll per unit leaf area was reduced considerably by salt stress, however. Stomatal conductance declined substantially with salt stress such that the intercellular CO2 concentration (Ci) was reduced by up to 30%. Salinization of plants was found to alter the δ13C value of leaves of Phaseolus by up to 5‰ and this change agreed quantitatively with that predicted by the theory relating carbon-isotope fractionation to the corresponding measured intercellular CO2 concentration. Salt stress also brought about a reduction in photosynthetic CO2 fixation independent of altered diffusional limitations. The initial slope of the photosynthesis versus Ci response declined with salinity stress, indicating that the apparent in-vivo activity of RuBP carboxylase was decreased by up to 40% at high leaf Cl- concentrations. The quantum yield for net CO2 uptake was also reduced by salt stress.

Osmotic adjustment and the inhibition of leaf, root, stem and silk growth at low water potentials in maize

Abstract: The expansion growth of plant organs is inhibited at low water potentials (Ψ w), but the inhibition has not been compared in different organs of the same plant Therefore, we determined elongation rates of the roots, stems, leaves, and styles (silks) of maize (Zea mays L) as soil water was depleted The Ψ w was measured in the region of cell expansion of each organ The complicating effects of transpiration were avoided by making measurements at the end of the dark period when the air had been saturated with water vapor for 10 h and transpiration was less than 1% of the rate in the light Growth was inhibited as the Ψ w in the region of cell expansion decreased in each organ The Ψ w required to stop growth was-050,-075, and-100 MPa, in this order, in the stem, silks, and leaves However, the roots grew at these Ψ w and ceased only when Ψ w was lower than-14 MPa The osmotic potential decreased in each region of cell expansion and, in leaves, roots and stems, the decrease was sufficient to maintain turgor fully In the silks, the decrease was less and turgor fell In the mature tissue, the Ψ w of the stem, leaves and roots was similar to that of the soil when adequate water was supplied This indicated that an equilibrium existed between these tissues, the vascular system, and the soil At the same time, the Ψ w was lower in the expanding regions than in the mature tissues, indicating that there was a Ψ w disequilibrium between the growing tissue and the vascular system The disequilibrium was interpreted as a Ψ w gradient for supplying water to the enlarging cells When water was withheld, this gradient disappeared in the leaf because Ψ w decreased more in the xylem than in the soil, indicating that a high flow resistance had developed in the xylem In the roots, the gradient did not decrease because vascular Ψ w changed about the same amount as the soil Ψ w Therefore, the gradient in Ψ w favored water uptake by roots but not leaves at low Ψ w The data show that expansion growth responds to low Ψ w differently in different growing regions of the plant Because growth depends on the maintenance of turgor for extending the cell walls and the presence of Ψ w gradients for supplying water to the expanding cells, several factors could have been responsible for these differences The decrease of turgor in the silks and the loss of the Ψ w gradient in the leaves probably contributed to the high sensitivity of these organs In the leaves, the gradient loss was so complete that it would have prevented growth regardless of other changes In the roots, the maintenance of turgor and Ψ w gradients probably allowed growth to continue This difference in turgor and gradient maintenance could contribute to the increase in root/shoot ratios generally observed in water-limited conditions

Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine

Abstract: The bud-inducing effect of the cytokinin N6-(Δ2-isopentenyl)-adenine (i6-Ade) was examined in the moss Physcomitrella patens growing in liquid culture. Under these conditions, buds could be induced on chloronemata as well as on caulonemata. By application of i6-Ade, bud-formation was accelerated in both types of tissue. The number of buds, their size and their site of development were dependent on the concentration of the cytokinin in the range of 10-7 M to 10-5 M. Moreover, the percentage of caulonema cells increased with a cytokinin concentration of 10-5 M. These results indicate that chloronema cells may also function as target cells for exogenous cytokinins. The composition of proteins from caulonemata and chloronemata of two different species (P. patens and Funaria hygrometrica), grown on solid medium were compared. No differences could be detected between the protein patterns of caulonemata and chloronemata of the same species while between the two species the differences were obvious.

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs.

Abstract: The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.

Effect of temperature and light on the toxicity and growth of the blue-green alga Microcystis aeruginosa (UV-006).

Abstract: The toxicity and growth of Microcystis aeruginosa (UV-006) from the Hartbeespoort Dam, South Africa were investigated at different temperatures and photon fluence rates under laboratory conditions. Cells harvested in late logarithmic growth phase were most toxic when grown at 20°C (LD50) median lethal dose [IP, mouse]=25.4 mg kg(-1)). Toxicity was markedly reduced at growth temperatures above 28° C. Fluence rate had a smaller effect on the toxicity of the cells, but toxicity tended to be less at the very low and high light fluences. Optimal conditions for growth did not coincide with those for toxin production. Well-aerated cultures of this isolate kept at pH 9.5 by CO2 addition, a temperature of 20-24° C, a fluence rate of 145 μmol photons m(-2) s(-1) and harvested in the late logarithmic growth phase yielded the maximum quantity of toxin.

Composition and function of plastoglobuli : II. Lipid composition of leaves and plastoglobuli during beech leaf senescence.

Abstract: The lipid composition of whole leaves and isolated plastoglobul of beech (Fagus sylvatica) has been studied during four natural autumnal senescence stages. Chlorophylls, glycolipids, and phospholipids were extensively degraded in leaves. About 20% of the glycolipids found in leaves during summer, however, remained in the last stage of leaf senescence. Triacylglycerols, also detected in large amounts in summer leaves, were hydrolyzed during senescence. The content of free fatty acids derived from degradation of glycerolipids therefore increased. The total carotenoid and prenyl quinone content was largely unchanged during senescence, except during the last stage investigated, but the reduced forms of prenyl quinones decreased while the oxidized prenyl quinones increased. Plastoglobuli isolated from summer leaves mainly contained triacylglycerols, plastohydroquinone, and α-tocopherol. The triacylglycerol content declined in plastoglobuli during senescence. Most of the triacylglycerols must be located outside the plastoglobuli throughout the stages investigated. Carotenoids liberated from thylakoids were esterified and increasingly deposited in plastoglobuli during senescence. In the last senescence stage, carotenoid esters were the main component of plastoglobuli. Prenyl quinones were also transferred into plastoglobuli. Reduced prenyl quinones were sucessively oxidized during senescence and plastoquinone (oxidized) was the predominant prenyl quinone in plastoglobuli isolated from the last senescence stage. The carotenoid and prenyl quinone distribution was identical in leaves and plastoglobuli during late senescence. The main constituents of thylakoids, glycolipids and proteins, were not deposited in plastoglobuli and therefore did not play an important role in plastoglobuli metabolism.

The cellular parameters of leaf development in tobacco: a clonal analysis.

Abstract: The cellular parameters of leaf development in tobacco (Nicotiana tabacum L.) have been characterized using clonal analysis, an approach that provides unequivocal evidence of cell lineage. Our results indicate that the tobacco leaf arises from a group of around 100 cells in the shoot apical meristem. Each of these cells contributes to a unique longitudinal section of the axis and transverse section of the lamina. This pattern of cell lincage indicates that primordial cells contribute more or less equally to the growth of the axis, in contrast to the more traditional view of leaf development in which the leaf is pictured as arising from a group of apical initials. Clones induced prior to the initiation of the lamina demonstrate that the subepidermal layer of the lamina arises from at least six files of cells. Submarginal cells usually divide with their spindles parallel to the margin, and therefore contribute relatively little to the transverse expansion of the lamina. During the expansion of the lamina the orientation and frequency of cell division are highly regulated, as is the duration of meristematic growth. Initially, cell division is polarized so as to produce lineages that are at an oblique angle to the midrib; later cell division is in alternating perpendicular planes. The distribution of clones generated by irradiation at various stages of development indicates that cell division ceases at the tip of the leaf when the leaf is about one tenth its final size, and then ceases in progressively more basal regions of the lamina. Variation in the mutation frequency within the lamina reflects variation in the frequency of mitosis. Prior to the mergence of the leaf the frequency of mutation is maximal near the tip of the leaf and extremely low at its base; after emergence, the frequency of mutation increases at the base of the leaf. In any given region of the lamina the frequency of mutation is highest in interveinal regions, and is relatively low near the margin. Thus, both the orientation and frequency of cell division at the leaf margin indicate that this region plays a minor role in the growth of the lamina.

The relationship between the redox state of Q A and photosynthesis in leaves at various carbon-dioxide, oxygen and light regimes.

Abstract: The response of chlorophyll fluorescence elicited by a low-fluence-rate modulated measuring beam to actinic light and to superimposed 1-s pulses from a high-fluence-rate light source was used to measure the redox state of the primary acceptor Q A of photosystem II in leaves which were photosynthesizing under steady-state conditions. The leaves were exposed to various O2 and CO2 concentrations and to different energy fluence rates of actinic light to assess the relationship between rates of photosynthesis and the redox state of Q A. Both at low and high fluence rates, the redox state of Q A was little altered when the CO2 concentration was reduced from saturation to about 600 μl·l(-1) although photosynthesis was decreased particularly at high fluence rates. Upon further reduction in CO2 content the amount of reduced Q A increased appreciably even at low fluence rates where light limited CO2 reduction. Both in the presence and in the absence of CO2, a more reduced Q A was observed when the O2 concentration was below 2%. Q A was almost fully reduced when leaves were exposed to high fluence rates under nitrogen. Even at low fluence rates, Q A was more reduced in shade leaves of Asarum europaeum and Fagus sylvatica than in leaves of Helianthus annuus and Fagus sylvatica grown under high light. Also, in shade leaves the redox state of Q A changed more during a transition from air containing 350 μl·l(-1) CO2 to CO2-free air than in sun leaves. The results are discussed with respect to the energy status and the CO2-fixation rate of the leaves.

Ethylene-induced microtubule reorientations: mediation by helical arrays.

Abstract: Entire microtubule arrays, within outer cortical and epidermal cells of pea epicotyl and mung-bean hypocotyl, have been visualized by indirect immunofluorescence. In all cells the microtubule arrangement can be interpreted as being a single multistart helix of variable pitch. In control cells the predominant pattern is a tightly compressed helix with the microtubules consequently in a net transverse direction with respect to the cell axis. Occasionally some cells show an oblique helix and rare cells show a longitudinal array which may be interpreted as a steeply pitched helix. By contrast in ethylene treated tissue, many cells show net longitudinal and oblique arrays of microtubules and few show transverse arrays. Similar effects can be induced by high osmolality. It is suggested that the plant cortical cytoskeleton is an integral unit, capable of wholesale reorientation in response to environmental signals.

The developmental morphology and growth dynamics of the tobacco leaf

Abstract: The developmental morphology and growth dynamics of the leaf of Nicotiana tabacum L. cv. Xanthi Nc. are described. Epidermal and internal cell patterns indicate that the leaf axis arises from approx. 100 cells in four layers of the shoot apex, while the lamina arises from several rows of cells in each of three layers of the leaf axis. Cell patterns at the apex and margin of the leaf do not support the classical view that these regions have a specialized meristematic function. Instead the development of the leaf appears to be largely dependent on intercalary growth. The pattern of growth within the lamina is surprisingly complex. In addition to a proximal-distal gradient in the duration of growth and cell division during development, localized transitory changes in the rate of these processes also occur. These observations are discussed in reference to previous discriptions of leaf development in tobacco.

Composition and function of plastoglobuli. I: Isolation and purification from chloroplasts and chromoplasts

Abstract: Plastoglobuli have been isolated and purified from chloroplasts of beech and spinach leaves and from broom flower chromoplasts by a repeated floating-gradient technique. The main components in plastoglobuli isolated from chloroplasts were triacylglycerols and lipophilic prenyl quinones, mainly plastohydroquinone and α-tocopherol. The corresponding oxidized prenyl quinones, plastoquinone (ox), α-tocoquinone, and the phylloquinone vitamin K1, were detected in trace amounts. Plastoglobuli isolated from chromoplasts contained large amounts of carotenoid esters. Triacylglycerols constituted two-thirds of the content of these plastoglobuli. The total prenyl quinone content was low in chromoplast plastoglobuli. Plastoquinone (ox) was the major prenyl quinone constituent. Plastoglobuli contained small amounts of chlorophylls, carotenoids (with the exception of chromoplast plastoglobuli), glycolipids, and proteins due to adsorption phenomena during the isolation process; however, increasing purification of the plastoglobuli fractions resulted in an exponential decline of these components. Adsorption of thylakoid lipids onto the plastoglobuli during the isolation process was demonstrated using an artificial globuli system. Therefore, pigments, glyco- and phospholipids, and proteins were regarded as thylakoid contaminations and not as actual constituents of plastoglobuli.

Photosynthesis and ribulose-1,5-bisphosphate carboxylase/oxygenase in rice leaves from emergence through senescence. Quantitative analysis by carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate

Abstract: Changes in gas-exchange rates during the life span of the leaves of rice (Oryza sativa L.) were analyzed quantitatively by measuring changes in the carboxylation/oxygenation and regeneration of ribulose 1,5-bisphosphate (RuBP) at photon fluence rates of 2000 (saturating) and 500 (subsaturating) μmol quanta·m-2·s-1 under ambient air conditions. The RuBP levels were always higher than the active-site concentrations of RuBP carboxylase (EC 4.1.1.39), irrespective of the irradiance supplied. Analysis of the CO2-assimilation rate as a function of intercellular CO2 concentration indicated that RuBP regeneration does not limit CO2 assimilation. The estimated RuBP-carboxylase/oxygenase activity in vivo was linearly correlated to the rate of CO2 assimilation at each level of irradiance. This enzyme activity was just enough to account for the rate of CO2 assimilation at the saturating irradiance and was 35% more than the rate of CO2 assimilation at the subsaturating irradiance. Analysis of the assimilation rate at subsaturating irradiance as a function of intercellular CO2 concentration indicated that a limitation caused by enzyme activation comes into play. The results indicate that the rate of CO2 assimilation in rice leaves under ambient air conditions is limited during their entire life span by the RuBP-carboxylation/oxygenation capacity.

Evidence against the acid-growth theory of auxin action.

Abstract: Four experimental predictions of the ‘acid-growth theory’ of auxin (indole-3-acetic acid, IAA) action in inducing cell elongation were reinvestigated using abraded segments of maize (Zea mays L.) coleoptiles. i) Quantitative comparison of segment elongation and medium-acidification kinetics measured in the same sample of tissue reveals that these IAA-induced processes are neither correlated in time nor responding coordinately to cations present in the medium. ii) Exogenous protons are not able to substitute for IAA in causing segment elongation at the predicted pH of 4.5–5.0. Instead, external buffers induce significant segment elongation only below pH 4.5, reaching a maximal response at pH 1.75–2.5. Acid and IAA coact additively, and therefore independently, in the whole range of feasible pH values. iii) Neutral or alkaline buffers (pH 6–10) are unable to abolish the IAA-mediated growth response and have no effect on its lag-phase. iv) Fusicoccin, at a concentration producing the same H+ excretion as high concentrations of IAA, is ineffective in inducing segment elongation. Moreover, sucrose and other sugars can quantiatively substritute for IAA in inducing H+ excretion but are likewise ineffective in inducing elongation. It is concluded that these results are incompatible with the acid-growth theory of auxin action.

Hydrogen-isotope composition of leaf water in C3 and C4 plants: its relationship to the hydrogen-isotope composition of dry matter

Abstract: The natural abundance hydrogen-isotope composition of leaf water (\(\delta _{\text{D}}^{{\text{H}}_{\text{2}} {\text{O}}} \)) and leaf organic matter (δDorg) was measured in leaves of C3 and C4 dicotyledons and monocotyledons. The \(\delta _{\text{D}}^{{\text{H}}_{\text{2}} {\text{O}}} \) value of leaf water showed a marked diurnal variation, greatest enrichment being observed about midday. However, this variation was greater in the more slowly transpiring C4 plants than in C3 plants under comparable environmental conditions. A model based on analogies with a constant feed pan of evaporating water was developed and the difference between C3 and C4 plants expressed in terms of either differences in kinetic enrichment or different leaf morphology. Microclimatic and morphological features of the leaves which may be associated with this factor are discussed. There was no daily excursion in the δDorg value in leaves of either C3 or C4 plants. When δDorg values were referenced to the mean \(\delta _{\text{D}}^{{\text{H}}_{\text{2}} {\text{O}}} \) values during the period of active photosynthesis, the discrimination against deuterium during photosynthetic metabolism (ΔD) was greater in C3 plants (-117 to -121‰) than in C4 plants (-86 to -109‰).

Salt tolerance in the halophyte Suaeda maritima L. Dum. : The maintenance of turgor pressure and water-potential gradients in plants growing at different salinities.

Abstract: Osmotic potentials and individual epidermal cell turgor pressures were measured in the leaves of seedlings of Suaeda maritima growing over a range of salinities. Leaf osmotic potentials were lower (more negative) the higher the salt concentration of the solution and were lowest in the youngest leaves and stem apices, producing a gradient of osmotic potential towards the apex of the plant. Epidermal cell turgor pressures were of the order of 0.25 to 0.3 MPa in the youngest leaves measured, decreasing to under 0.05 MPa for the oldest leaves. This pattern of turgor pressure was largely unaffected by external salinity. Calculation of leaf water potential indicated that the gradient between young leaves and the external medium was not altered by salinity, but with older leaves, however, this gradient diminished from being the same as that for young leaves in the absence of NaCl, to under 30% of this value at 400 mM NaCl. These results are discussed in relation to the growth response of S. maritima.

Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.

Abstract: A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (Mr) of 118000 and a lesser-abundant 124000-Mr polypeptide. Under nondenaturing conditions all of the 124000-Mr species is immunoprecipitable, but the 118000-Mr species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-Mr species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.

Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen.

Abstract: Adventitious roots of two to four-weekold intact plants of Zea mays L. (cv. LG11) were shorter but less dense after extending into stagnant, non-aerated nutrient solution than into solution continuously aerated with air. Dissolved oxygen in the non-aerated solutions decreased from 21 kPa to 3–9 kPa within 24 h. When oxygen partial pressures similar to those found in non-aerated solutions (3, 5 and 12 kPa) were applied for 7 d to root systems growing in vigorously bubbled solutions, the volume of gas-space in the cortex (aerenchyma) was increased several fold. This stimulation of aerenchyma was associated with faster ethylene production by 45-mm-long apical root segments. When ethylene production by roots exposed to 5 kPa oxygen was inhibited by aminoethoxyvinylglycine (AVG) dissolved in the nutrient solution, aerenchyma formation was also retarded. The effect of AVG was reversible by concomitant applications of 1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene. Addition of silver nitrate, an inhibitor of ethylene action, to the nutrient solution also prevented the development of aerenchyma in roots given 5 kPa oxygen. Treating roots with only 1 kPa oxygen stimulated ethylene production but failed to promote gas-space formation. These severely oxygen-deficient roots seemed insensitive to the ethylene produced since a supplement of exogeneous ethylene that promoted aerenchyma development in nutrient solution aerated with air (21 kPa oxygen) failed to do so in nutrient solution supplied with 1 kPa oxygen. Both ethylene production and aerenchyma formation were almost completely halted when roots were exposed to nutrient solutions devoid of oxygen. Thus both processes require oxygen and are stimulated by oxygen-deficient surroundings in the 3-to 12-kPa range of oxygen partial pressures when compared with rates observed in air (21 kPa oxygen).

Control of the rate of cell enlargement: Excision, wall relaxation, and growth-induced water potentials

Abstract: A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential (Ψ s ), turgor pressure (Ψ p ) could be calculated. In the intact plant, Ψ s and Ψ p were essentially constant for the entire 22 h measurement, but Ψ s was lower and Ψ p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, Ψ s and Ψ p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue Ψ p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The Ψ p decreased because the cell walls relaxed as extension, caused by Ψ p , continued briefly without water uptake. The Ψ p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or Ψ p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in Ψ p.

Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow.

Abstract: Lucifer yellow has been microinjected into stomatal cells of Allium cepa L epidermal slices and Commelina communis L epidermal peels and the symplastic spread of dye to neighboring cells monitored by fluorescence microscopy Dye does not move out of injected mature guard cells, nor does it spread into the guard cells when adjacent epidermal or subsidiary cells are injected Dye does spread from injected subsidiary cells to other subsidiary cells These results are consistent with the reported absence of plasmodesmata in the walls of mature guard cells Microinjection was also used to ascertain when dye coupling ceases during stomatal differentiation in Allium Dye rapidly moves into and out of guard mother cells and young guard cells Hovewer, dye movement ceases midway through development as the guard cells begin to swell but well before a pore first opens Since plasmodesmata are still present at this stage, the loss of symplastic transport may result from changes in these structures well in advance of their actual disappearance from the guard cell wall

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Abstract: Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.

Isolation and characterization of Populus isoperoxidases involved in the last step of lignin formation.

Abstract: Peroxidases (EC 1.11.1.7) from Populus x euramericana were investigated during the dormant and growing seasons using histochemical and biochemical methods. The activities of syringaldazine oxidase and p-paraphenylenediamine-pyrocatechol oxidase in sections of branches were maximal during spring in both phloem and young xylem. Cytoplasmic and cell-wall peroxidase activities from different lignified tissues were estimated in vitro. Pronounced differences were noticed between fractions isolated during spring and winter. Gel electrophoresis showed the presence of an anionic fast-migrating isoperoxidase group with a high syringaldazine-oxidase activity. The isoenzymes of this group were different in winter and in spring. The properties of these isoperoxidases (kinetic constants, pH optimum, resistance to heat) were investigated after isolation by ion-exchange chromatography.

The intercellular compartmentation of metabolites in leaves of Zea mays L.

Abstract: Sap extracted from attached leaves of two-to three-week-old maize plants witt the aid of a roller device was almost devoid of bundle-sheath contamination as judged by the distribution of mesophyll and bundle-sheath markers. The extraction could be done very rapidly (less than 1 s) and the extract immediately quenched in HClO4 or reserved for enzyme assay. Comparison of the contents of metabolites in intact leaves and in the leaf extract allowed estimation of the distribution of metabolites between the bundle-sheath and the mesophyll compartments. Substantial amounts of metabolites such as malate and amino acids were present in the non-photosynthetic cells of the midrib. In the illuminated leaf, triose phosphate was predominantly located outside the bundle-sheath while the major part of the 3-phosphoglycerate was in the bundle sheath. The results indicate the existence of concentration gradients of triose phosphate and 3-phosphoglycerate in the leaf which are capable of maintaining carbon flow between the mesophyll and bundle-sheath cells during photosynthesis. There was no evidence for the existence of a gradient of pyruvate between the bundle-sheath and the mesophyll cells.

Simultaneous and independent effects of abscisic acid on stomata and the photosynthetic apparatus in whole leaves

Abstract: (±)-Abscisic acid (ABA) at 10-5 M was added to the transpiration stream of leaves of 16 species (C3 and C4, monocotyledons and dicotyledons). Stomatal responses followed one of three patterns: i) stomata that were wide and insensitive to CO2 initially, closed partially and became sensitive to CO2; ii) for stomata that were sensitive to CO2 before the application of ABA, the range of highest sensitivity to CO2 shifted from high to low intercellular partial pressures of CO2, for instance in leaves of Zea mays from 170–350 to 70–140 μbar; iii) when stomata responded strongly to ABA, their conductance was reduced to a small fraction of the initial conductance, and sensitivity to CO2 was lost. The photosynthetic apparatus was affected by applications of ABA to various degrees, from no response at all (in agreement with several previous reports on the absence of effects of ABA on photosynthesis) through a temporary decrease of its activity to a lasting reduction. Saturation curves of photosynthesis with respect to the partial pressure of CO2 in the intercellular spaces indicated that application of ABA could produce three phenomena: i) a reduction of the initial slope of the saturation curve (which indicates a diminished carboxylation efficiency); ii) a reduction of the level of the CO2-saturated rate of assimilation (which indicates a reduction of the ribulose-1,5-bisphosphate regeneration capacity); and iii) an increase of the CO2 compensation point. Photosynthesis of isolated mesophyll cells was not affected by ABA treatments. Responses of the stomatal and photosynthetic apparatus were usually synchronous and often proportional to each other, with the result that the partial pressure of CO2 in the intercellular spaces frequently remained constant in spite of large changes in conductance and assimilation rate. Guard cells and the photosynthetic apparatus were able to recover from effects of ABA applications while the ABA supply continued. Recovery was usually partial, in the case of the photosynthetic apparatus occasionally complete. Abscisic acid did not cause stomatal closure or decreases in the rate of photosynthesis when it was applied during a phase of stomatal opening and induction of photosynthesis that followed a transition from darkness to light.

Effects of drought on photosynthesis, chlorophyll fluorescence and photoinhibition susceptibility in intact willow leaves

Abstract: Plants from clonal cuttings of Salix sp. were subjected to a drying cycle of 10 d in a controlled environment. Gas exchange and fluorescence emission were measured on attached leaves. The light-saturated photosynthetic CO2 uptake became progressively inhibited with decreased leaf water potential both at high, and especially, at low intercellular CO2 pressure. The maximal quantum yield of CO2 uptake was more resistant. The inhibition of light-saturated CO2 uptake at leaf water potentials around-10 bar, measured at a natural ambient CO2 concentration, was equally attributable to stomatal and non-stomatal factors, but the further inhibition below this water-stress level was caused solely by non-stomatal factors. The kinetics of fluorescence emission was changed at severe water stress; the slow secondary oscillations of the induction curve were attenuated, and this probably indicates perturbations in the carbon reduction cycle. The influence of light level during the drought period was also studied. Provided the leaves were properly light-acclimated, drought at high and low light levels produced essentially the same effects on photosynthesis. However, low-light-acclimated leaves became more susceptible to photoinhibitory treatment under severe water stress, as compared with well-watered conditions.

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum.

Abstract: The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.

Day-night changes in leaf water relations associated with the rhythm of crassulacean acid metabolism in Kalanchoë daigremontiana

Abstract: A study was made of the day-night changes under controlled environmental conditions in the bulk-leaf water relations of Kalanchoe daigremontiana, a plant showing Crassulacean acid metabolism. In addition to nocturnal stomatal opening and net CO2 uptake, the leaves of well-watered plants showed high rates of gas exchange during the whole of the second part of the light period. Measurements with the pressure chamber showed that xylem tension increased during the night and then decreased towards a minimum at about midday; a significant increase in xylem tension was also seen in the late afternoon. Cell-sap osmotic pressure paralleled leaf malate content and was maximum at dawn and minimum at dusk. The relationship between these two variables indicated that the nocturnally synthesized malate was apparently behaving as an ideal osmoticum. To estimate bulk-leaf turgor pressure, values for water potential were derived by correcting the pressurechamber readings for the osmotic pressure of the xylem sap. This itself was found to depend on the malate content of the leaves. Bulk-leaf turgor pressure changed rhythmically during the day-night cycle; turgor was low during the late afternoon and for most of the night, but increased quickly to a maximum of 0.20 MPa around midday. In water-stressed plants, where net CO2 uptake was restricted to the dark period, there was also an increase in bulk-leaf turgor pressure at the start of the light period, but of reduced magnitude. Such changes in turgor pressure are likely to be of considerable ecological importance for the water economy of crassulacean-acid-metabolism plants growing in their natural habitats.

Structure and function of the microtubular cytoskeleton during pollen development in Gasteria verrucosa (Mill.) H. Duval

Abstract: In a study of pollen development in Gasteria verrucosa, the changes in the spatial organization of microtubules were related to the processes of cell division, nuclear movement and cytomorphogenesis. Sections of polyethylene-glycol-embedded anthers of G. verrucosa were processed immunocytochemically to record the structure and succession of fluorescently labeled microtubular configurations. Using microspectrophotometric measurements the relative quantity of tubulin in microtubules per unit of cytoplasm was determined. Cell dimensions and nuclear positions were measured to relate changes in cell shape and nuclear movements to microtubular configurations. Microtubules were detected in the different cells during microsporogenesis and microgametogenesis. In microspore mother cells which are approximately isodiametric at interphase, microtubules were predominantly arranged in a criss-cross pattern. The microtubules probably function as a flexible cytoskeleton which sustains the integrity of the cytoplasm. Bundles of microtubules were observed in the microspores, in the generative cells and during nuclear division, where they functioned in establishing and maintaining cell and spindle shapes. Microtubules radiating from nuclear membranes appeared to fix the nucleus in position. In prophase of meiosis and after microspore mitosis, periods a high fluorescence intensity were distinguished indicating a variation in the quantity of microtubules.