Showing papers in "Planta in 1991"

Partitioning of photosynthetic electron flow between CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.) at different CO2 concentrations and during drought stress

Abstract: Photosystem II chlorophyll fluorescence and leaf net gas exchanges (CO2 and H2O) were measured simultaneously on bean leaves (Phaseolus vulgaris L.) submitted either to different ambient CO2 concentrations or to a drought stress. When leaves are under photorespiratory conditions, a simple fluorescence parameter ΔF/ Fm (B. Genty et al. 1989, Biochem. Biophys. Acta 990, 87-92; ΔF = difference between maximum, Fm, and steady-state fluorescence emissions) allows the calculation of the total rate of photosynthetic electron-transport and the rate of electron transport to O2. These rates are in agreement with the measurements of leaf O2 absorption using (18)O2 and the kinetic properties of ribulose-1,5bisphosphate carboxylase/oxygenase. The fluorescence parameter, ΔF/Fm, showed that the allocation of photosynthetic electrons to O2 was increased during the desiccation of a leaf. Decreasing leaf net CO2 uptake, either by decreasing the ambient CO2 concentration or by dehydrating a leaf, had the same effect on the partitioning of photosynthetic electrons between CO2 and O2 reduction. It is concluded that the decline of net CO2 uptake of a leaf under drought stress is only due, at least for a mild reversible stress (causing at most a leaf water deficit of 35%), to stomatal closure which leads to a decrease in leaf internal CO2 concentration. Since, during the dehydration of a leaf, the calculated internal CO2 concentration remained constant or even increased we conclude that this calculation is misleading under such conditions.

Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex

Abstract: Several chitin-binding proteins were isolated from the “bottom fraction” of Hevea brasiliensis (Mull.) Arg. latex. One of these chitin-binding proteins is hevein, a small monomeric protein which strongly resembles the lectin from stinging nettle (Urtica dioica L.). Like the latter, hevein showed strong antifungal activity against several fungi in vitro. The possible involvement of this protein in the defense against invasion by potentially pathogenic fungi is discussed.

Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of plasma-membrane H(+)-ATPase.

Abstract: Auxin causes elongation growth of plant cells by increasing the plastic extensibility of the cell wall. Putative cellular events involved in this hormone action were studied using maize (Zea mays L.) coleoptiles with the following results: (i) Auxin enhances membrane flow from the endoplasmic reticulum to the plasma membrane (PM). This effect was demonstrated by pulse-labeling of the endoplasmic reticulum with myo-[3H]inositol in coleoptile segments and by measuring the distribution of the label within isolated and separated microsomal membrane fractions, (ii) Auxin rapidly increases the amount of antibody-detectable H+-ATPase in the PM. This augmentation is already significant 10 min after the addition of indole-3-acetic acid (IAA) and reaches a new higher steady-state level after about 30 min. (iii) Cycloheximide, a potent inhibitor of both protein synthesis and extension growth, quickly diminishes the auxin-enhanced level of the PM H+-ATPase, indicating an apparent half-life of the enzyme of around 12 min. (iv) Cordycepin, which blocks the synthesis of mRNAs, reduces the auxin-elevated level of the H+-ATPase similar to cycloheximide. (v) Changes in the growth rate of coleoptile segments in response to IAA, cycloheximide, and cordycepin exactly reflect the changes of the H+-ATPase level in the PM. (vi) The elongation growth induced by fusicoccin, or ester compounds, or by an elevated CO2 concentration in the incubation medium, is not related to an increased number of H+-ATPase molecules within the PM. (vii) The necessity of H+ for cell-wall-loosening processes is again demonstrated by growth experiments with abraded coleoptile segments. The adjustment of the cell wall to a pH of ≥6.5 completely abolishes the auxin-induced elongation growth; no inhibition occurs with non-abraded segments. Buffer solutions of pH ≤6.0 induce “acid growth” of abraded segments for several hours. It is suggested that auxin activates a cluster of genes responsible (i) for the induction and acceleration of exocytotic processes (e.g. by the synthesis of either proteins, necessary for the fusion of membranes, or of other effectors); (ii) for the synthesis of PM H+-ATPases, increasing the capacity for H+-extrusion into the apoplast as a precondition for wall enlargement (“acid growth”); (iii) for a supposed synthesis and exocytosis of certain proteins, enzymes and wall precursors necessary for wall metabolism and the “repair” of the proton-loosened and turgor-stretched cell wall. Both, fusicoccin and auxin affect cell-wall plasticity according to the “acid-growth” theory. However, the mechanisms leading to this event are completely different; the auxinenhanced H+-extrusion is a gene-controlled process.

Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Abstract: Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl−, K+ and Ca2+ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.

Decreased ribulose-1,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with "antisense" rbcS : I. Impact on photosynthesis in ambient growth conditions.

Abstract: Experiments were carried out to determine how decreased expression of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) affects photosynthetic metabolism in ambient growth conditions. In a series of tobacco (Nicotiana tabacum L.) plants containing progressively smaller amounts of Rubisco the rate of photosynthesis was measured under conditions similar to those in which the plants had been grown (310 μmol photons · m(-2) · s(-1), 350 μbar CO2, 22° C). (i) There was only a marginal inhibition (6%) of photosynthesis when Rubisco was decreased to about 60% of the amount in the wildtype. The reduced amount of Rubisco was compensated for by an increase in Rubisco activation (rising from 60 to 100%), with minor contributions from an increase of its substrates (ribulose-1,5-bisphosphate and the internal CO2 concentration) and a decrease of its product (glycerate-3-phosphate). (ii) The decreased amount of Rubisco was accompanied by an increased ATP/ADP ratio that may be causally linked to the increased activation of Rubisco. An increase of highenergy-state chlorophyll fluorescence shows that thylakoid membrane energisation and high-energy-state-dependent energy dissipation at photosystem two had also increased. (iii) A further decrease of Rubisco (in the range of 50-20% of the wildtype level) resulted in a strong and proportional inhibition of CO2 assimilation. This was accompanied by a decrease of fructose-1,6-bisphosphatase activity, coupling-factor 1 (CF1)-ATP-synthase protein, NADP-malate dehydrogenase protein, and chlorophyll. The chlorophyll a/b ratio did not change, and enolase and sucrose-phosphate synthase activity did not decrease. It is argued that other photosynthetic enzymes are also decreased once Rubisco decreases to the point at which it becomes strongly limiting for photosynthesis. (iv) It is proposed that the amount of Rubisco in the wildtype represents a balance between the demands of light, water and nitrogen utilisation. The wildtype overinvests about 15% more protein in Rubisco than is needed to avoid a strict Rubisco limitation of photosynthesis. However, this "excess" Rubisco allows the wildtype to operate with lower thylakoid energisation, and decreased high-energy-state-dependent energy dissipation, hence increasing light-use efficiency by about 6%. It also allows the wildtype to operate with a lower internal CO2 concentration in the leaf and a lower stomatal conductance at a given rate of photosynthesis, so that instantaneous water-use efficiency is marginally (8%) increased.

Temperature dependence of violaxanthin de-epoxidation and non-photochemical fluorescence quenching in intact leaves of Gossypium hirsutum L. and Malva parviflora L.

Abstract: The temperature dependence of the rate of de-epoxidation of violaxanthin to zeaxanthin was determined in leaves of chilling-sensitive Gossypium hirsutum L. (cotton) and chilling-resistant Malva parviflora L. by measurements of the increase in absorbance at 505 nm (ΔA 505) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between ΔA 505 and the decrease in the epoxidation state of the xanthophyll-cycle pigment pool was obtained over the range 10-40° C. The maximal rate of de-epoxidation was strongly temperature dependent; Q10 measured around the temperature at which the leaf had developed was 2.1-2.3 in both species. In field-grown Malva the rate of de-epoxidation at any given measurement temperature was two to three times higher in leaves developed at a relatively low temperature in the early spring than in those developed in summer. Q10 measured around 15° C was in the range 2.2-2.6 in both kinds of Malva leaves, whereas it was as high as 4.6 in cotton leaves developed at a daytime temperature of 30° C. Whereas the maximum (initial) rate of de-epoxidation showed a strong decrease with decreased temperature the degree of de-epoxidation reached in cotton leaves after a 1-2 · h exposure to a constant photon flux density increased with decreased temperature as the rate of photosynthesis decrease. The zeaxanthin content rose from 2 mmol · (mol chlorophyll)(-1) at 30° C to 61 mmol · (mol Chl)(-1) at 10° C, corresponding to a de-epoxidation of 70% of the violaxanthin pool at 10° C. The degree of de-epoxidation at each temperature was clearly related to the amount of excessive light present at that temperature. The relationship between non-photochemical quenching of chlorophyll fluorescence and zeaxanthin formation at different temperatures was determined for both untreated control leaves and for leaves in which zeaxanthin formation was prevented by dithiothreitol treatment. The rate of development of that portion of non-photochemical quenching which was inhibited by dithiothreitol decreased with decreasing temperature and was linearly related to the rate of zeaxanthin formation over a wide temperature range. In contrast, the rate of development of the dithiothreitol-resistant portion of non-photochemical quenching was remarkably little affected by temperature. Evidently, the kinetics of the development of non-photochemical quenching upon exposure of leaves to excessive light is therefore in large part determined by the rate of zeaxanthin formation. For reasons that remain to be determined the relaxation of dithiothreitolsensitive quenching that is normally observed upon darkening of illuminated leaves was strongly inhibited at low temperatures.

Ribulose-1,5-bisphosphate carboxylase-oxygenase, other Calvin-cycle enzymes, and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream.

Abstract: The inhibition of photosynthesis after supplying glucose to detached leaves of spinach (Spinacia oleracea L.) was used as a model system to search for mechanisms which potentially contribute to the “sink” regulation of photosynthesis. Detached leaves were supplied with 50 mM glucose or water for 7 d through the transpiration stream, holding the leaves in low irradiance (16 μmol photons · m−2 · s−1) and a cycle of 9 h light/15 h darkness to prevent any endogenous accumulation of carbohydrate. Leaves supplied with water only showed marginal changes of photosynthesis, respiration, enzyme levels or metabolites. When leaves were supplied with 50 mM glucose, photosynthesis was gradually inhibited over several days. The inhibition was most marked when photosynthesis was measured in saturating irradiance and ambient CO2, less marked in saturating irradiance and saturating CO2, and least marked in limiting irradiance. There was a gradual loss of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein, fructose-1,6-bisphosphatase, NADP-glyceraldehyde-3-phosphate dehydrogenase and chlorophyll. The inhibition of photosynthesis was accompanied by a large decrease of glycerate-3-phosphate, an increase of triose-phosphates and fructose-1,6-bisphospate, and a small decrease of ribulose-1,5-bisphosphate. The stromal NADPH/NADP ratio increased (as indicated by increased activation of NADP-malate dehydrogenase), and the ATP/ADP ratio increased. Chlorophyll-fluorescence analysis indicated that thylakoid energisation was increased, and that the acceptor side of photosystem II was more reduced. Similar results were obtained when glucose was supplied by floating leaf discs in low irradiance on glucose solution, and when detached spinach leaves were held in high light to produce an endogenous accumulation of carbohydrate. Feeding glucose also led to an increased rate of respiration. This was not accompanied by any changes of pyruvate kinase, phosphofructokinase, or pyrophosphate: fructose-6-phosphate phosphotransferase activity. There was a decrease of phosphoenolpyruvate, glycerate-3-phosphate and glycerate-2-phosphate, an increase of pyruvate and triose-phosphates, and an increased ATP/ADP ratio. These results show (i) that accumulation of carbohydrate can inhibit photosynthesis via a long-term mechanism involving a decrease of Rubisco and other Calvin-cycle enzymes and (ii) that respiration is stimulated due to an unknown mechanism, which increases the utilisation of phosphoenolpyruvate.

Sink Regulation of Photosynthetic Metabolism in Transgenic Tobacco Plants Expressing Yeast Invertase in Their Cell-Wall Involves a Decrease of the Calvin-Cycle Enzymes and an Increase of Glycolytic-Enzymes

Abstract: Leaves on transgenic tobacco plants expressing yeast-derived invertase in the apoplast develop clearly demarcated green and bleached sectors when they mature. The green areas contain low levels of soluble sugars and starch which are turned over on a daily basis, and have high rates of photosynthesis and low rates of respiration. The pale areas accumulate carbohydrate, photosynthesis is inhibited, and respiration increases. This provides a model system to investigate the “sink” regulation of photosynthetic metabolism by accumulating carbohydrate. The inhibition of photosynthesis is accompanied by a decrease of ribulose-1,5-bisphosphate and glycerate-3-phosphate, and an increase of triosephosphate and fructose-1,6-bisphosphate. The extracted activities of ribulose-1,5-bisphosphate carboxylase, fructose-1, 6-bisphosphatase and NADP-glyeraldehyde-3-phosphate dehydrogenase decreased. The activity of sucrose-phosphate synthase remained high or increased, an increased portion of the photosynthate was partitioned into soluble sugars rather than starch, and the pale areas showed few or no oscillations during transitions between darkness and saturating light in saturating CO2. The increased rate of respiration was accompanied by an increased level of hexose-phosphates, triose-phosphates and fructose-1,6-bisphosphate while glycerate-3-phosphate and phosphoenolpyruvate decreased and pyruvate increased. The activities of pyruvate kinase, phosphofructokinase and pyrophosphate: fructose-6-phosphate phosphotransferase increased two- to four-fold. We conclude that an increased level of carbohydrate leads to a decreased level of Calvin-cycle enzymes and, thence, to an inhibition of photosynthesis. It also leads to an increased level of glycolytic enzymes and, thence, to a stimulation of respiration. These changes of enzymes are more important in middle- or long-term adjustments to high carbohydrate levels in the leaf than fine regulation due to depletion of inorganic phosphate or high levels of phosphorylated metabolites.

The location of (1-3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1-3)-β-glucan-specific monoclonal antibody

Abstract: The location of the (1→3)-β-glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (1→3)-β-glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (1→3, 1→4)-β-glucan-BSA conjugate. Binding was inhibited by (1→3)-β-oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (1→3)-β-linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 104M−1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (1→3)-β-glucan in the inner wall layer of thin sections of the N. alata pollen tubes.

Functioning of photosystems I and II in pea leaves exposed to heat stress in the presence or absence of light Analysis using in-vivo fluorescence, absorbance, oxygen and photoacoustic measurements

Abstract: Fluorimetric, photoacoustic, polarographic and absorbance techniques were used to measure in situ various functional aspects of the photochemical apparatus of photosynthesis in intact pea leaves (Pisum sativum L.) after short exposures to a high temperature of 40 ° C. The results indicated (i) that the in-vivo responses of the two photosystems to high-temperature pretreatments were markedly different and in some respects opposite, with photosystem (PS) II activity being inhibited (or down-regulated) and PSI function being stimulated; and (ii) that light strongly interacts with the response of the photosystems, acting as an efficient protector of the photochemical activity against its inactivation by heat. When imposed in the dark, heat provoked a drastic inhibition of photosynthetic oxygen evolution and photochemical energy storage, correlated with a marked loss of variable PSII-chlorophyll fluorescence emission. None of the above changes were observed in leaves which were illuminated during heating. This photoprotection was saturated at rather low light fluence rates (around 10 W · m−2). Heat stress in darkness appeared to increase the capacity for cyclic electron flow around PSI, as indicated by the enhanced photochemical energy storage in far-red light and the faster decay of P 700 + (oxidized reaction center of PSI) monitored upon sudded interruption of the far-red light. The presence of light during heat stress reduced somewhat this PSI-driven cyclic electron transport. It was also observed that heat stress in darkness resulted in the progressive closure of the PSI reaction centers in leaves under steady illumination whereas PSII traps remained largely open, possibly reflecting the adjustment of the photochemical efficiency of undamaged PSI to the reduced rate of photochemistry in PSII.

Photoacclimation and photoinhibition in Ulva rotundata as influenced by nitrogen availability.

Abstract: Clonal tissue of the marine chlorophyte macroalga, Ulva rotundata Blid., was transferred from 100 to 1700 μmol photons · m−2 · s−1 under limiting (1.5 μM NH 4 + maximum, N/P=2) and sufficient (15 μM NH 4 + maximum, N/P=20) nitrogen supply at 18° C and 11 h light-13 h darkness daily. Photoinhibition was assayed by light-response curves (photosynthetic O2 exchange), and chlorophyll fluorescence at 77 K and room temperature. Daily surface-area growth rate (μSA) in N-sufficient plants increased sixfold over 3 d and was sustained at that level. During this period, respiration (R d) doubled and light-saturated net photosynthesis capacity (P m) increased by nearly 50%, indicating acclimation to high light. Quantum yield (ϕ) decreased by 25% on the first day, but recovered completely within one week. The ratio of variable to maximum fluorescence (F v/F m) also decreased markedly on the first day, because of an increase in initial fluorescence (F o) and a decrease in F m, and partially recovered over several days. Under the added stress of N deficiency, μSA accelerated fivefold over 4 d, despite chronic photoinhibition, then declined along with tissue-N. Respiration doubled, but P m decreased by 50% over one week, indicating inability to acclimate to high light. Both ϕ and F v/F m decreased markedly on the first day and did not significantly recover. Changes in F o, F m and xanthophyll-cycle components indicate concurrent photodamage to photosystem II (PSII) and photoprotection by thermal deexcitation in the antenna pigments. Increasing μSA coincided with photoinhibition of PSII. Insufficient diel-carbon balance because of elevated R d and declining P m and tissue-N, rather than photochemical damage per se, was the apparent proximate cause of decelerating growth rate and subsequent tissue degeneration under N deficiency in U. rotundata.

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts.

Abstract: A functional assay for gibberellin (GA) receptors is described based on the induction of α-amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA4 immobilised to Sepharose beads. A 17-thiol derivative of GA4, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA4-17-Sepharose induces high levels of α-amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA4. In order to eliminate the possibility that GA4 may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA4, GA4-17-Sepharose and control Sepharose estimated by determining the relative amounts of α-amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA417-Sepharose induces α-amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling.

Abstract: A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, 1H-, 13C-nuclear magnetic resonance and mass spectrometry as α-linolenic acid. When applied to detached tendrils, exogenous α-linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of α-linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40–80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4–5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.

Decreased Ribulose-1,5-Bisphosphate Carboxylase-Oxygenase in Transgenic Tobacco Transformed with Antisense Rbcs. 2. Flux-Control Coefficients for Photosynthesis in Varying Light, Co2, and Air Humidity

Abstract: Transgenic tobacco (Nicotiana tabacum L.) plants transformed with 'antisense' rbcS to produce a series of plants with a progressive decrease in the amount of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been used to investigate the contribution of Rubsico to the control of photosynthesis at different irradiance, CO2 concentrations and vapour-pressure deficits. Assimilation rates, transpiration, the internal CO2 concentration and chlorophyll fluorescence were measured in each plant. (i) The flux-control coefficient of Rubisco was estimated from the slope of the plot of Rubisco content versus assimilation rate. The flux-control coefficient had a value of 0.8 or more in high irradiance, (1050 μmol·m(-2)·s(-1)), low-vapour pressure deficit (4 mbar) and ambient CO2 (350 μbar). Control was marginal in enhanced CO2 (450 μbar) or low light (310 μmol·m(-2)·s(-1)) and was also decreased at high vapour-pressure deficit (17 mbar). No control was exerted in 5% CO2. (ii) The flux-control coefficients of Rubisco were compared with the fractional demand placed on the calculated available Rubisco capacity. Only a marginal control on photosynthetic flux is exerted by Rubisco until over 50% of the available capacity is being used. Control increases as utilisation rises to 80%, and approaches unity (i.e. strict limitation) when more than 80% of the available capacity is being used. (iii) In low light, plants with reduced Rubisco have very high energy-dependent quenching of chlorophyll fluorescence (qE) and a decreased apparent quantum yield. It is argued that Rubisco still exerts marginal control in these conditions because decreased Rubisco leads to increased thylakoid energisation and high-energy dependent dissipation of light energy, and lower light-harvesting efficiency. (iv) The flux-control coefficient of stomata for photosynthesis was calculated from the flux-control coefficient of Rubisco and the internal CO2 concentration, by applying the connectivity theorem. Control by the stomata varies between zero and about 0.25. It is increased by increased irradiance, decreased CO2 or decreased vapour-pressure deficit. (v) Photosynthetic oscillations in saturating irradiance and CO2 are suppressed in decreased-activity transformants before the steady-state rate of photosynthesis is affected. This provides direct evidence that these oscillations reveal the presence of "excess" Rubisco. (vi) Comparison of the flux-control coefficients of Rubisco with mechanistic models of photosynthesis provides direct support for the reliability of these models in conditions where Rubisco has a flux-control coefficient approach unity (i.e. "limits" photosynthesis), but also indicates that these models are less useful in conditions where control is shared between Rubisco and other components of the photosynthetic apparatus.

A "futile" cycle of sucrose synthesis and degradation is involved in regulating partitioning between sucrose, starch and respiration in cotyledons of germinating Ricinus communis L. seedlings when phloem transport is inhibited.

Abstract: There was a dramatic alteration in the pattern of metabolism of [U14C]glucose by cotyledons of germinating Ricinus communis L. seedlings when phloem transport was inhibited by removing most of the hypocotyl and root. (i) Incorporation into sucrose was decreased two- to threefold, incorporation into starch was stimulated three- to sixfold, and there was a small increase of respiration, (ii) Pulse-chase experiments using 14C and measurements of the total sucrose content revealed a rapid cycle of sucrose synthesis and degradation. When export is inhibited there is a two- to threefold inhibition of unidirectional sucrose synthesis and a three-fold stimulation of unidirectional sucrose degradation. As a result, the net flux switches from rapid net synthesis to slow net mobilisation of sucrose, (iii) The cotyledons contained adequate activities of sucrose synthase, acid and alkaline invertase and sucrose-phosphate synthase to catalyse the observed rate of sucrose breakdown and synthesis, respectively. The extracted activities of the degradative enzymes did not change after inhibiting phloem transport. The maximum activity of sucrose-phosphate synthase was also unaltered, but the activity measured in the presence of limiting substrates and phosphate was decreased twofold, indicating that sucrose-phosphate synthase has been deactivated by a mechanism analogous to that occurring in spinach leaves. (iv) The switch from sucrose export to starch synthesis when phloem transport was prevented was accompanied by only a small (20–50%) increase of the sucrose concentration in the cotyledons, no change of hexose-phosphates, an increase (16–70%) of fructose-1,6-bisphosphate and triosephosphate, and a small decrease (15–30%) of glycerate-3-phosphate, glycerate-2-phosphate and phosphoenolpyruvate. Fructose-2,6-bisphosphate and pyrophosphate doubled when 10 mM phosphate was included in the medium bathing the cotyledons, but not when phosphate was omitted (v) It is concluded that a futile cycle involving simultaneous synthesis and degradation of sucrose allows sucrose metabolism to respond in an extremely sensitive manner when phloem export is inhibited. There is a dramatic switch of flux through the sucrose pool, even though there are only marginal changes in the concentrations of sucrose and metabolites, or in the rate of respiration.

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures : 1. The kinetics of alkaloid production and the influence of feeding intermediate metabolites.

Abstract: The factors by which the endogenous regulation of tropane-alkaloid biosynthesis may be effected have been examined in a transformed root culture of Datura stramonium. Pools of intermediates showed a subculture-related maximal accumulation, as did the enzyme activities by which they are synthesised and/or metabolised. The end-products, principally hyoscyamine and apohyoscyamine, in contrast, accumulated steadily in growing cultures. Feeding putrescine, agmatine or tropine did not enhance alkaloid accumulation, but rather may even have resulted in a lowering of hyoscyamine levels. Similarly, feeding precursors for the tropate moiety of hyoscyamine either had no influence or had a detrimental effect on hyoscyamine accumulation. Under some feeding conditions, intermediates in the pathway from N-methylputrescine up to and including tropine accumulated up to 40-fold. Little effect on early intermediates was found, however, when tropinone or tropine were fed. The expression of the enzyme arginine decarboxylase (EC 4.1.1.19) was particularly sensitive to feed-back repression, both by its product agmatine and by more distant pathway intermediates, notably putrescine and tropine. Some diminution of the levels of putrescine N-methyltransferase (EC 2.1.1.53) and N-methylputrescine oxidase, the first committed enzymes of alkaloid biosynthesis, was also seen with tropine, although only at rather high levels. It is concluded that the pathway is not regulated in a simple manner and that (i) the early enzymes of the pathway are at near rate-limiting levels, (ii) there is a major limitation to flux at the level of the esterification of tropine, and (iii) the level of free tropine may be important in determining the flux into and through the tropane pathway.

Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.

Abstract: Three-week-old protonemata of Funaria hygrometrica Hedw. cultivated in Petri dishes tolerate slow drying (24 h to complete dryness) but not rapid drying (1h to complete dryness). Slowly dried mosses show, on a dry-weight basis, a sixfold increase in abscisic-acid (ABA) contents during the drying process. Rehydrated, slowly dried protonemata have the ability to tolerate subsequent rapid drying. When ABA is added to three-week-old protonemata at a concentration of 10 μM for 16 h, tolerance to rapid drying is induced. These data indicate that the induction of drought tolerance in Funaria hygrometrica is mediated by ABA. Mosses treated with ABA loose their water as fast as controls do; therefore, ABA does not act via reduced water loss. However, induction of synthesis of new proteins by ABA may form an important part of the drought tolerance because 10 μM cycloheximide inhibits the ABA-mediated tolerance to rapid drying.

A Chlamydomonas reinhardtii low-starch mutant is defective for 3-phosphoglycerate activation and orthophosphate inhibition of ADP-glucose pyrophosphorylase

Abstract: A low-starch mutant accumulating less than 5% of wild-type amounts was isolated after X-ray mutagenesis of Chlamydomonas reinhardtii cells. The recessive st-1-1 defect segregated as a single mendelian mutation through meiosis, and led to a severe decrease in starch accumulation under all culture conditions tested, whether in the light or in darkness. Adenosine 5′-diphosphoglucose pyrophosphorylase (in the absence of 3-phosphoglycerate), starch synthase, phosphoglucomutase, phosphorylase and starch-branching enzyme were all characterized and shown to be unaffected by the mutation. However, ADP-glucose pyrophosphorylase in the mutant had its sensitivity to activation by 3-phosphoglycerate lowered dramatically and became less responsive to orthophosphate. Our results are consistent both with a mutation in a structural gene of a multisubunit enzyme or in a regulatory gene responsible for switching ADP-glucose pyrophosphorylase from a 3-phosphoglycerate-insensitive to a 3-phosphoglycerate-sensitive form. These results provide definite proof of the in-vivo requirement for 3-phosphoglycerate activation to obtain substantial starch synthesis in plants. The conclusions hold both for synthesis from CO2 in the light or from exogenous organic carbon sources in darkness. A model is presented in which the existence of a 3-phosphoglycerate gradient explains localized starch synthesis around the pyrenoid of lower plants.

Tunicamycin-inhibited carrot somatic embryogenesis can be restored by secreted cationic peroxidase isoenzymes

Abstract: Somatic embryogenesis of carrot (Daucus carota L.) is inhibited by the glycosylation inhibitor tunicamycin. This inhibition is reversible by the addition of correctly glycosylated glycoproteins which have been secreted into the culture medium. To identify the proteins responsible for complementation, glycoproteins present in the medium of embryo cultures were purified and tested for their activity in the tunicamycin inhibition/ complementation assay. A 38-kDa glycoprotein was purified that could restore embryogenesis to more than 50% of that in untreated controls. This 38-kDa glycoprotein was identified as a heme-containing peroxidase on the basis of its A405/A280 ratio (Reinheit Zahl or RZ) and enzyme activity. The 38-kDa peroxidase consisted of four different cationic isoenzymes of which only one or possibly two appeared active in the complementation assay. The cationic peroxidase isoenzymes from the carrot medium could be effectively replaced by cationic horseradish peroxidases which depended on their catalytic properties for their ability to restore tunicamycin-inhibited somatic embryogenesis.

A comparative study into the chemical constitution of cutins and suberins from Picea abies (L.) Karst., Quercus robur L., and Fagus sylvatica L.

Abstract: The compositions of BF3/CH3OH depolymerisates of cutins and suberins from leaf and periderm samples from Picea abies [L.] Karst., Quercus robur L., and Fagus sylvatica L., respectively, were determined by quantitative capillary gas chromatography/mass spectroscopy. Long-chain monobasic, ω-hydroxymonobasic, dihydroxymonobasic, trihydroxymonobasic and epoxyhydroxymonobasic alkanoic acids constituted the major aliphatic monomers of leaf cutins. The total amounts of cutin monomers ranged from 629 mg · m−2 (Fagus) to 1350 mg · m−2 (Quercus). Cutin composition and amounts did not significantly differ between current year and three-year-old needles of Picea. Trans-esterification of periderm samples yielded a much greater variety of aliphatic monomers than obtained from cutins. In addition to the substance classes found with cutins, suberin depolymerisates also contained α, ω-dibasic acids while dihydroxymonobasic acids were lacking. Depolymerisates from periderms taken from different locations on a Picea tree did not differ significantly in their relative composition. The results are discussed in terms of the distinctive characteristics of the aliphatic portions of cutins and suberins, respectively. Discriminant analysis is applied for formulating a quantitative and inarbitrary classification rule for cutins and suberins. The precision, statistical significance and robustness of this classification rule are tested by employing it to a large set of compositional data (70 plant species) from the literature. The relevance of data obtained by depolymerization methods for elucidating the physical structure of cutins and suberins in situ is evaluated.

Prediction of photoinhibition of photosynthesis from measurements of fluorescence quenching components

Abstract: Photoinhibition of photosynthesis in willow (Salix sp.) leaves was investigated by measuring the ratio of variable (Fv) to maximal (FM) chlorophyll fluorescence. The use of this parameter was justified as it showed similar kinetics of recovery from photoinhibition as did the light-limited rate of gross photosynthesis. When leaves of different status were exposed to different environmental conditions for 1 h, the large variation in photoinhibition subsequently observed was not correlated with the incident photosynthetic photon flux density. However, it was positively correlated with the fraction of closed reaction centres of photosystem (PS) II, and negatively correlated with the magnitude of the high-energy fluorescence quenching, both of which were measured during the treatment. The best correlation was obtained when the two parameters were taken together in a multiple regression model. Young high-light-acclimated leaves were the most resistant to photoinhibition at a given fraction of closed PSII centres, which was explained by these having the most prominent high-energy quenching. Leaves illuminated in the presence of dithiothreitol and the uncoupler nigericin were found at the other end of the correlation. However, the markedly lowered high-energy quenching induced by dithiothreitol was not fully matched by the expected increase in photoinhibition. The fraction of closed PSII centres provided a relative not an absolute measure of excessive excitation, since the correlation model varied depending on the length of the treatment. In a separate experiment the response to fluctuating light under otherwise constant conditions was studied. It was found that under these more restricted conditions photoinhibition could be predicted from the integrated light dose.

Computer-based video digitizer analysis of surface extension in maize roots: kinetics of growth rate changes during gravitropism

Abstract: We used a video digitizer system to measure surface extension and curvature in gravistimulated primary roots of maize (Zea mays L.). Downward curvature began about 25 +/- 7 min after gravistimulation and resulted from a combination of enhanced growth along the upper surface and reduced growth along the lower surface relative to growth in vertically oriented controls. The roots curved at a rate of 1.4 +/- 0.5 degrees min-1 but the pattern of curvature varied somewhat. In about 35% of the samples the roots curved steadily downward and the rate of curvature slowed as the root neared 90 degrees. A final angle of about 90 degrees was reached 110 +/- 35 min after the start of gravistimulation. In about 65% of the samples there was a period of backward curvature (partial reversal of curvature) during the response. In some cases (about 15% of those showing a period of reverse bending) this period of backward curvature occurred before the root reached 90 degrees. Following transient backward curvature, downward curvature resumed and the root approached a final angle of about 90 degrees. In about 65% of the roots showing a period of reverse curvature, the roots curved steadily past the vertical, reaching maximum curvature about 205 +/- 65 min after gravistimulation. The direction of curvature then reversed back toward the vertical. After one or two oscillations about the vertical the roots obtained a vertical orientation and the distribution of growth within the root tip became the same as that prior to gravistimulation. The period of transient backward curvature coincided with and was evidently caused by enhancement of growth along the concave and inhibition of growth along the convex side of the curve, a pattern opposite to that prevailing in the earlier stages of downward curvature. There were periods during the gravitropic response when the normally unimodal growth-rate distribution within the elongation zone became bimodal with two peaks of rapid elongation separated by a region of reduced elongation rate. This occurred at different times on the convex and concave sides of the graviresponding root. During the period of steady downward curvature the elongation zone along the convex side extended farther toward the tip than in the vertical control. During the period of reduced rate of curvature, the zone of elongation extended farther toward the tip along the concave side of the root. The data show that the gravitropic response pattern varies with time and involves changes in localized elongation rates as well as changes in the length and position of the elongation zone. Models of root gravitropic curvature based on simple unimodal inhibition of growth along the lower side cannot account for these complex growth patterns.

Involvement of cinnamyl-alcohol dehydrogenase in the control of lignin formation in Sorghum bicolor L. Moench.

Abstract: The lignin structure and enzyme activities of normal and brown-midrib (BMR-6) mutant lines of Sorghum bicolor have been compared to identify the enzyme(s) involved in the reduction of the lignin content of the mutant. The results indicate that cinnamyl-alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase are depressed in the BMR-6 line, whereas the structural modifications correspond only to a reduction of CAD activity. Apparently, the change in the Sorghum lignin content, caused by depression of CAD activity, is accompanied by the incorporation of cinnamaldehydes into the core lignin.

Messenger-RNA and protein changes associated with induction of Brassica microspore embryogenesis

Abstract: Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.

Induction of stilbene synthase by Botrytis cinerea in cultured grapevine cells

Abstract: The interaction between Botrytis cinerea Pers. and grapevine (Vitis vinifera L.) was studied in a model system of reduced complexity. Cultured plant cells and fragments of fungal cell wall were used to simulate some of the processes taking place upon infection of grapevine with B. cinerea. A soluble glucan elicitor was prepared from the fungal cell wall by acid hydrolysis. Like the insoluble wall preparation, the soluble fragment derived from the cell wall acted upon plant cells in eliciting stilbene formation. In grapevine cells, the interaction with the fungus led to a dramatic shut-off general protein synthesis and to the selective formation of a small set of proteins involved in induced resistance. The proteins synthesized de novo with highest rates were stilbene synthase (StiSy) and l-phenylalanine ammonia-lyase (PAL). Stilbene synthase was purified to apparent homogeneity and its molecular properties were characterized. The enzyme is a homodimer with subunit Mr 43 000 and pl = 5.4. Although there were indications of the presence of isoenzymes, these were not distinguished by charge differences. In size, the grapevine StiSy shows microheterogeneity and differs from the appreciably larger enzyme prepared from peanut. Prior to induction by fungal attack, virtually no stilbenes are formed in the plant cell. Upon induction of the pathway leading to the stilbene resveratrol, StiSy activity determines the ratelimiting step in the metabolic sequence. The highly induced grapevine cells produce and secrete resveratrol and derivatives which are known to be fungistatic.

Sucrose storage in cell suspension cultures of Saccharum sp. (sugarcane) is regulated by a cycle of synthesis and degradation.

Abstract: We have investigated the regulation of sucrose storage in cell-suspension cultures of sugarcane. When grown in batch culture, sucrose accumulation commences after about 5 d, when the nitrogen supply is exhausted. Sucrose storage is also induced by decreasing the nitrogen supply to cells growing in a chemostat. The measured activity of sucrose-phosphate synthase is high enough to account for the rate of sucrose accumulation, provided precautions are taken to avoid the hydrolysis of UDP during the assay. The cells contained high sucrose-synthase activity but pulsing experiments with [14C]glucose and unlabelled fructose indicated that this enzyme did not contribute substantially to the synthesis of sucrose, because the glucosyl and fructosyl moieties of sucrose were equally labelled. Several lines of evidence demonstrate the presence of a cycle in which sucrose is synthesized and degraded simultaneously; sucrosephosphate-synthase activity doubles during the phase when the cells are actively storing sucrose but activity is also high after storage has ceased, or when the sucrose is being remobilised; pulse experiments with [14C]fructose also showed that sucrose synthesis occurs not only during the storage phase, but also after storage has stopped and during the rapid mobilisation of sucrose; the cells contain high activities of sucrose synthase and alkaline invertase and these are both at a maximum when sucrose storage is occurring; even during the storage phase. [14C]fructose pulses lead to labelling of free glucose which is evidence for rapid synthesis and degradation of sucrose. It is proposed that the rate and extent of sucrose storage is regulated by this cycle of synthesis and degradation. Measurements of enzyme activities and metabolite levels are presented, and it is discussed which factors could contribute to the regulation of these two opposing fluxes and, hence, the rate of net sucrose storage and mobilisation.

Compartmental nitrate concentrations in barley root cells measured with nitrate-selective microelectrodes and by single-cell sap sampling

Abstract: Nitrate-selective microelectrodes were used to measure intracellular nitrate concentrations (as activities) in epidermal and cortical cells of roots of 5-d-old barley (Hordeum vulgare L.) seedlings grown in nutrient solution containing 10 mol · m−3 nitrate. Measurements in each cell type grouped into two populations with mean (±SE) values of 5.4 ± 0.5 mol · m−3 (n=19) and 41.8 ± 2.6 mol · m−3 (n = 35) in epidermal cells, and 3.2 ± 1.2 mol · m−3 (n = 4) and 72.8 ± 8.4 mol · m−3 (n = 13) in cortical cells. These could represent the cytoplasmic and vacuolar nitrate concentrations, respectively, in each cell type. To test this hypothesis, a single-cell sampling procedure was used to withdraw a vacuolar sap sample from individual epidermal and cortical cells. Measurement of the nitrate concentration in these samples by a fluorometric nitrate-reductase assay confirmed a mean vacuolar nitrate concentration of 52.6 ± 5.3 mol · m−3 (n = 10) in epidermal cells and 101.2 ± 4.8 mol · m−3 (n = 44) in cortical cells. The nitrate-reductase assay gave only a single population of measurements in each cell type, supporting the hypothesis that the higher of the two populations of electrode measurements in each cell type are vacuolar in origin. Differences in the absolute values obtained by these methods are probably related to the fact that the nitrate electrodes were calibrated against nitrate activity but the enzymic assay against concentration. Furthermore, a 28-h time course for the accumulation of nitrate measured with electrodes in epidermal cells showed the apparent cytoplasmic measurements remained constant at 5.0 ± 0.7 mol · m−3, while the vacuole accumulated nitrate to 30–50 mol · m−3. The implications of the data for mechanisms of nitrate transport at the plasma membrane and tonoplast are discussed.

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos

Abstract: The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following 14C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.

The microtubular cytoskeleton during development of the zygote, proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) heynh

Abstract: The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm in A. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition.

Abstract: Factors influencing the rate of superoxide (O 2 (-) ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O 2 (-) )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O 2 (-) production by thylakoids, incubated at 25° C and 350 μmol photon · m(-2) · s(-1) (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 μmol · (mg chlorophyll)(-1) · h(-1). Incubation at 5° C and a moderate PFD, decreased the rate of O 2 (-) production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O 2 (-) -production at 25° C by 75-100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O 2 (-) production. In the absence of added electron acceptors, thylakoids produced O 2 (-) at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea reduced O 2 (-) production under all conditions tested. The results show that the rate of O 2 (-) production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O 2 (-) production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.