Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (I. Light-Harvesting Complex II Abundance and Zeaxanthin Content in Chlorella vulgaris)

The basis of the increased resistance to photoinhibition upon growth at low temperature was investigated. Photosystem II (PSII) excitation pressure was estimated in vivo as 1 - qp (photochemical quenching). We established that Chlorella vulgaris exposed to either 5[deg]C/150 [mu]mol m-2 s-1 or 27[deg]C/2200 [mu]mol m-2 s-1 experienced a high PSII excitation pressure of 0.70 to 0.75. In contrast, Chlorella exposed to either 27[deg]C/150 [mu]mol m-2 s-1 or 5[deg]C/20 [mu]mol m-2 s-1 experienced a low PSII excitation pressure of 0.10 to 0.20. Chlorella grown under either regime at high PSII excitation pressure exhibited: (a) 3-fold higher light-saturated rates of O2 evolution; (b) the complete conversion of PSII[alpha] centers to PSII[beta] centers; (c) a 3-fold lower epoxidation state of the xanthophyll cycle intermediates; (d) a 2.4-fold higher ratio of chlorophyll a/b; and (e) a lower abundance of light-harvesting polypeptides than Chlorella grown at either regime at low PSII excitation pressure. In addition, cells grown at 5[deg]C/150 [mu]mol m-2 s-1 exhibited resistance to photoinhibition comparable to that of cells grown at 27[deg]C/2200 [mu]mol m-2 s-1 and were 3- to 4-fold more resistant to photoinhibition than cells grown at either regime at low excitation pressure. We conclude that increased resistance to photoinhibition upon growth at low temperature reflects photosynthetic adjustment to high excitation pressure, which results in an increased capacity for nonradiative dissipation of excess light through zeaxanthin coupled with a lower probability of light absorption due to reduced chlorophyll per cell and decreased abundance of light-harvesting polypeptides.     

Photosystem II Excitation Pressure and Development of Resistance to Photoinhibition (II. Adjustment of Photosynthetic Capacity in Winter Wheat and Winter Rye)

Winter wheat (Triticum aestivum L. cv Monopol), spring wheat (Triticum aestivum L. cv Katepwa), and winter rye (Secale cereale L. cv Musketeer) grown at 5[deg]C and moderate irradiance (250 [mu]mol m-2 s-1) (5/250) exhibit an increased tolerance to photoinhibition at low temperature in comparison to plants grown at 20[deg]C and 250 [mu]mol m-2 s-1 (20/250). However, 5/250 plants exhibited a higher photosystem II (PSII) excitation pressure (0.32-0.63) than 20/250 plants (0.18-0.21), measured as 1 - qP, the coefficient of photochemical quenching. Plants grown at 20[deg]C and a high irradiance (800 [mu]mol m-2 s-1) (20/800) also exhibited a high PSII excitation pressure (0.32-0.48). Similarly, plants grown at 20/800 exhibited a comparable tolerance to photoinhibition relative to plants grown at 5/250. In contrast to a recent report for Chlorella vulgaris (D.P. Maxwell, S. Falk, N.P.A. Huner [1995] Plant Physiol 107: 687-694), this tolerance to photoinhibition occurs in winter rye with minimal adjustment to polypeptides of the PSII light-harvesting complex, chlorophyll a/b ratios, or xanthophyll cycle carotenoids. However, Monopol winter wheat exhibited a 2.5-fold stimulation of sucrosephosphate synthase activity upon growth at 5/250, in comparison to Katepwa spring wheat. We demonstrate that low-temperature-induced tolerance to photoinhibition is not a low-temperature-growth effect per se but, instead, reflects increased photosynthetic capacity in response to elevated PSII excitation pressure, which may be modulated by either temperature or irradiance.     

Redox Regulation of Light-Harvesting Complex II and cab mRNA Abundance in Dunaliella salina

We demonstrate that photosynthetic adjustment at the level of the light-harvesting complex associated with photosystem II (LCHII) in Dunaliella salina is a response to changes in the redox state of intersystem electron transport as estimated by photosystem II (PSII) excitation pressure. To elucidate the molecular basis of this phenomenon, LHCII apoprotein accumulation and cab mRNA abundance were examined. Growth regimes that induced low, but equivalent, excitation pressures (either 13[deg]C/20 [mu]mol m-2 s-1 or 30[deg]C/150 ([mu]mol m-2 s-1) resulted in increased LHCII apoprotein and cab mRNA accumulation relative to algal cultures grown under high excitation pressures (either 13[deg]C/150 [mu]mol m-2 s-1 or 30[deg]C/2500 [mu]mol m-2 s-1). Thermodynamic relaxation of high excitation pressures, accomplished by shifting cultures from a 13 to a 30[deg]C growth regime at constant irradiance for 12 h, resulted in a 6- and 8-fold increase in LHCII apoprotein and cab mRNA abundance, respectively. Similarly, photodynamic relaxation of high excitation pressure, accomplished by a shift from a light to a dark growth regime at constant temperature, resulted in a 2.4- to 4-fold increase in LHCII apoprotein and cab mRNA levels, respectively. We conclude that photosynthetic adjustment to temperature mimics adjustment to high irradiance through a common redox sensing/signaling mechanism. Both temperature and light modulate the redox state of the first, stable quinone electron acceptor of PSII, which reflects the redox poise of intersystem electron transport. Changes in redox poise signal the nucleus to regulate cab mRNA abundance, which, in turn, determines the accumulation of light-harvesting apoprotein. This redox mechanism may represent a general acclimation mechanism for photosynthetic adjustment to environmental stimuli.     

Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.     

Effects of Growth Temperature on the Responses of Ribulose-1,5-Biphosphate Carboxylase,Electron Transport Components,and Sucrose Synthesis Enzymes to Leaf Nitrogen in Rice,and Their Relationships to Photosynthesis

Effects of growth temperature on the photosynthetic gas-exchange rates and their underlying biochemical properties were examined in young, fully expanded leaves of rice (Oryza sativa L.). The plants were grown hydroponically under day/night temperature regimes of 18/15[deg]C, 23/18[deg]C, and 30/23[deg]C and all photosynthetic measurements were made at a leaf temperature of 25[deg]C and an irradiance of 1800 [mu]mol quanta m-2 s-1. Growth temperature affected the photosynthetic CO2 response curve. The relative ratio of the initial slope to the CO2-saturated photosynthesis increased with rising growth temperature. This was caused mainly by an increase in CO2-limited photosynthesis for a given leaf nitrogen content with rising growth temperature. However, there was no difference in ribulose-1,5-bisphosphate carboxylase (Rubisco) content at any given leaf nitrogen content among temperature treatments. In addition, the activation state and catalytic turnover rate of Rubisco were not affected by growth temperature. The increase in CO2-limited photosynthesis with rising growth temperature was the result of an increase in the CO2 transfer conductance between the intercellular airspaces and the carboxylation sites. The amounts of total chlorophyll and light-harvesting chlorophyll a/b protein II increased for the same leaf nitrogen content with rising growth temperature, but the amounts of cytochrome f and coupling factor 1 and the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were the same between plants grown at 23/18[deg]C and those grown at 30/23[deg]C. Similarly, CO2-saturated photosynthesis was not different for the same leaf nitrogen content between these treatments. For the 18/15[deg]C-grown plants, a slight decrease in the amounts of cytochrome f and coupling factor 1 and an increase in the activities of cytosolic fructose-1,6-bisphosphatase and sucrose-phosphate synthase were found, but these were not reflected in CO2-saturated photosynthesis.     

Chloroplast Movement in the Shade Plant Tradescantia albiflora Helps Protect Photosystem II against Light Stress

The role of high-light-induced chloroplast movement in the photoprotection of the facultative shade plant Tradescantia albiflora was investigated by comparison with pea (Pisum sativum L.) leaves, both grown in 50 [mu]mol photons m-2 s-1. Photoinactivation of photosystem II (PSII) in vivo was induced in 1.1% CO2 by varying either duration (0-2 h) of illumination (fixed at 1800 [mu]mol m-2 s-1) or irradiance (0-3000 [mu]mol m-2 s-1) at a fixed duration (1 h) after infiltration of leaves with water or lincomycin (an inhibitor of chloroplast-encoded protein synthesis). At all photon exposures, PSII of T. albiflora leaves showed a greater resistance to light stress than pea leaves, although both utilization of absorbed light by photosynthesis and psbA gene product synthesis were smaller than for pea leaves. This greater tolerance was not due to differences in PSII antenna size or the index of susceptibility of PSII to light stress, because these two parameters were comparable in both plants. However, the transmittance increase mediated by chloroplast movement was greater in T. albiflora than pea, resulting in a 10% decrease of absorbed light at high light. We suggest that the greater tolerance of PSII against light stress in T. albiflora may be partly ascribed to its light-induced chloroplast rearrangement.     

Antenna Size Dependency of Photoinactivation of Photosystem II in Light-Acclimated Pea Leaves

Utilization of absorbed light energy by photosystem (PS) II for O2 evolution depends on the light-harvesting antenna size, but the role of antenna size in the photoinactivation of PSII seems controversial. To address this controversy, pea (Pisum sativum L.) plants were grown in low (50 [mu]mol m-2 s-1) or high (650 [mu]mol m-2 s-1) light. The doubled functional antenna size of PSII in low light allows each PSII to utilize twice as many photons at given flash light energies for O2 evolution. The application of a target theory to depict the photon dose dependency of PSII photoinactivation measured by repetitive-flash O2 yield and the ratio of variable to maximal chlorophyll fluorescence indicates that photoinactivation of PSII is probably a single-hit process in which repair or photoprotective mechanisms are only slightly involved. Furthermore, the exacerbation of photoinactivation of PSII with greater antenna size under anaerobic conditions strongly indicates that photoinactivation of PSII depends on antenna size.     

Sugar and Organic Acid Accumulation in Guard Cells of Vicia faba in Response to Red and Blue Light

Changes in neutral sugar and organic acid content of guard cells were quantitated by high-performance liquid chromatography during stomatal opening in different light qualities. Sonicated Vicia faba epidermal peels were irradiated with 10 [mu]mol m-2 s-1 of blue light, a fluence rate insufficient for the activation of guard cell photosynthesis, or 125 [mu]mol m-2 s-1 of red light, in the presence of 1 mM KCl, 0.1 mM CaCl2. The low-fluence-rate blue light stimulated an average net stomatal opening of 4.7 [mu]m in 2 h, whereas the saturating fluence rate of red light stimulated an average net opening of 3.8 [mu]m in 2 h. Under blue light, the malate content of guard cells increased to 173% of the initial level during the first 30 min of opening and declined as opening continued. Sucrose levels continuously rose throughout the blue light-stimulated opening, reaching 215% of the initial level after 2 h. The starch hydrolysis products maltose and maltotriose remained elevated at all times. Under red light, guard cells showed very little increase in organic acid or maltose levels, whereas sucrose levels increased to 208% of the initial level after 2 h. Total measured organic metabolite concentrations were correlated with stomatal apertures in all cases except where substantial malate increases occurred. These results support the hypothesis that light quality modulates alternative mechanisms of osmotic accumulation in guard cells, including potassium uptake, photosynthetic sugar production, and starch breakdown.     

Photosystem II Reaction Center Damage and Repair in Dunaliella salina (Green Alga) (Analysis under Physiological and Irradiance-Stress Conditions)

Mechanistic aspects of the photosystem II (PSII) damage and repair cycle in chloroplasts were investigated. The D1/32-kD reaction center protein of PSII (known as the psbA chloroplast gene product) undergoes a frequent light-dependent damage and turnover in the thylakoid membrane. In the model organism Dunaliella salina (green alga), growth under a limiting intensity of illumination (100 [mu]mol of photons m-2 s-1; low light) entails damage, degradation, and replacement of D1 every about 7 h. Growth under irradiance-stress conditions (2000 [mu]mol of photons m-2 s-1; high light) entails damage to and replacement of D1 about every 20 min. Thus, the rate of damage and repair of PSII appears to be proportional to the light intensity during plant growth. Low-light-grown cells do not possess the capacity for high rates of repair. Upon transfer of low-light-grown cells to high-light conditions, accelerated damage to reaction center proteins is followed by PSII disassembly and aggregation of neighboring reaction center complexes into an insoluble dimer form. The accumulation of inactive PSII centers that still contain the D1 protein suggests that the rate of D1 degradation is the rate-limiting step in the PSII repair cycle. Under irradiance-stress conditions, chloroplasts gradually acquire a greater capacity for repair. The induction of this phenomenon occurs with a half-time of about 24 h.     

Temperature Dependence of the Linkage of Quantum Yield of Photosystem II to CO2 Fixation in C4 and C3 Plants

The temperature dependence of quantum yields of electron transport from photosystem II (PSII) ([phi]II, determined from chlorophyll a fluorescence) and CO2 assimilation ([phi]CO2, apparent quantum yield for CO2 assimilation) were determined simultaneously in vivo. With C4 species representing NADP-malic enzyme, NAD-malic enzyme, and phosphoenolpyruvate carboxykinase subgroups, the ratio of [phi]II/[phi]CO2 was constant over the temperature range from 15 to 40[deg]C at high light intensity (1100 [mu]mol quanta m-2 s-1). A similar response was obtained at low light intensity (300 [mu]mol quanta m-2 s-1), except the ratio of [phi]II/[phi]CO2 increased at high temperature. When the true quantum yield for CO2 fixation ([phi]CO2*) was calculated by correcting for respiration in the light (estimated from temperature dependence of dark respiration), the ratio of [phi]II/[phi]C02* remained constant with varying temperature and under both light intensities in all C4 species examined. Because the [phi]II/[phi]CO2* ratio was the same in C4 monocots representing the three subgroups, the ratio was not affected by differences in the bio-chemical mechanism of concentrating CO2 in the bundle sheath cells. The results suggest that PSII activity is closely linked to the true rate of CO2 fixation in C4 plants. The close relationship between [phi]II and [phi]CO2* in C4 species under varying temperature and light intensity conditions is apparently due to a common low level of photorespiration and a primary requirement for reductive power in the C3 pathway. In contrast, in a C3 plant the [phi] II/[phi]CO2* ratio is higher under normal atmospheric conditions than under nonphotorespiratory conditions and it increases with rising temperature. This decrease in efficiency in utilizing energy derived from PSII for CO2 fixation is due to an increase in photorespiration. In both the C3 and C4 species, photochemistry is limited under low temperature, and thus excess energy must be dissipated by nonphotochemical means.     

Light Stress and Oxidative Cell Damage in Photoautotrophic Cell Suspension of Euphorbia characias L

A photoautotrophic cell-suspension culture of Euphorbia characias L. grown at 70 [mu]mol photons m-2 s-1 was very sensitive to light stress: the gross photosynthesis measured by using a mass spectrometric 16O2/18O2 isotope technique showed a fast decrease at a rather low light intensity of 100 [mu]mol photons m-2 s-1, far below the photosynthetic saturation level. The contribution of activated oxygen species on photosystem II photoinhibition was examined for a given light intensity. A protective effect on gross photosynthesis was observed with 1% oxygen. When light stress was applied to a methyl viologen-adapted cell suspension, photoinhibition was reduced. When 50 [mu]mol L-1 methyl viologen was added, photoinhibition was slightly enhanced. These responses suggested an involvement of superoxide radicals in the photoinhibition process of E. characias photoautotrophic cells. The long-term (16 h) effects of photoinhibition were then studied. Aldehyde (malondialdehyde and 4-hydroxyalcenals) production resulting from lipid peroxidation was stimulated in long-term stressed cells. When 50 [mu]mol L-1 methyl viologen were added, increased aldehyde production was measured. Under 1% oxygen, the aldehyde production was comparable to that of nonstressed cells. The relationship among lipid peroxidation, light intensity, and net photosynthesis suggests that aldehyde production may result from cell death provoked by a prolonged energy deficit due to the inhibition of photosynthesis.     

The Effect of Elevated [CO2] on Growth and Photosynthesis of Two Eucalyptus Species Exposed to High Temperatures and Water Deficits

Two species of eucalyptus (Eucalyptus macrorhyncha and Eucalyptus rossii) were grown for 8 weeks in either ambient (350 [mu]L L-1) or elevated (700 [mu]L L-1) CO2 concentrations, either well watered or without water additions, and subjected to a daily, 3-h high-temperature (45[deg]C, maximum) and high-light (1250 [mu]mol photons m-2 s-1, maximum) stress period. Water-stressed seedlings of E. macrorhyncha had higher leaf water potentials when grown in elevated [CO2]. Growth analysis indicated that increased [CO2] may allow eucalyptus species to perform better during conditions of low soil moisture. A down-regulation of photosynthetic capacity was observed for seedlings grown in elevated [CO2] when well watered but not when water stressed. Well-watered seedlings grown in elevated [CO2] had lower quantum efficiencies as measured by chlorophyll fluorescence (the ratio of variable to maximal chlorophyll fluorescence [Fv/Fm]) than seedlings grown in ambient [CO2] during the high-temperature stress period. However, no significant differences in Fv/Fm were observed between CO2 treatments when water was withheld. The reductions in dark-adapted Fv/Fm for plants grown in elevated [CO2] were not well correlated with increased xanthophyll cycle photoprotection. However, reductions in the Fv/Fm were correlated with increased levels of nonstructural carbohydrates. The reduction in quantum efficiencies for plants grown in elevated [CO2] is discussed in the context of feedback inhibition of electron transport associated with starch accumulation and variation in sink strength.     

Regulation of Photosynthetic Induction State by the Magnitude and Duration of Low Light Exposure

This study was undertaken to examine the dependence of the regulatory enzymes of photosynthetic induction on photon flux density (PFD) exposure in soybean (Glycine max L.). The induction state varies as a function of both the magnitude and duration of the PFD levels experienced prior to an increase in PFD. The photosynthetic induction state results from the combined activity of separate processes that each in turn depend on prior PFD environment in different ways. Direct measurement of enzyme activities coupled with determination of in situ metabolite pool sizes indicated that the fast-induction component was associated with the activation state of stromal fructose-1,6-bisphosphatase (FBPase, EC 3.1.3.11) and showed rapid deactivation in the dark and at low PFD. The fast-induction component was activated at low PFD levels, around 70 [mu]mol photons m-2 s-1. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 2.7.1.19) deactivated very slowly in the dark and required higher PFD for activation. Both enzymes saturated at lower PFD than did photosynthesis, around 400 [mu]mol photons m-2 s-1. Ribulose-5-phosphate kinase (EC 2.7.1.19) appeared never to be limiting to photosynthesis, and saturated at much lower PFD than either FBPase or Rubisco. Determination of photosynthetic metabolite pool sizes from leaves at different positions within a soybean canopy showed a limitation to carbon uptake at the stromal FBPase and possibly the sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37) in shade leaves upon initial illumination at saturating PFD levels.     

Photoacclimation of Prochlorococcus sp. (Prochlorophyta) Strains Isolated from the North Atlantic and the Mediterranean Sea

Two Atlantic (SARG and NATL1) strains and one Mediterranean (MED) strain of Prochlorococcus sp., a recently discovered marine, free-living prochlorophyte, were grown over a range of "white" irradiances (lg) and under low blue light to examine their photoacclimation capacity. All three strains contained divinyl (DV) chlorophylls (Chl) a and b, both distinguishable from "normal" Chls by their red-shifted blue absorption maximum, a Chl c-like pigment at low concentration, zeaxanthin, and [alpha]-carotene. The presence of two phaeophytin b peaks in acidified extracts from both Atlantic strains grown at high lg suggests that these strains also had a normal Chl b-like pigment. In these strains, the total Chl b to DV-Chl a molar ratio decreased from about 1 at 7.5 [mu]mol quanta m-2 s-1 to 0.4 to 0.5 at 133 [mu]mol quanta m-2 s-1. In contrast, the MED strain always had a low DV-Chl b to DV-Chl a molar ratio, ranging between 0.13 at low lg and 0.08 at high lg. The discrepancies between the Atlantic and MED strains could result from differences either in the number of light-harvesting complexes (LHC) II per photosystem II or in the Chl b-binding capacity of the apoproteins constituting LHC II. Photosynthesis was saturated at approximately 5 fg C(fg Chl)-1 h-1 or 6 fg C cell-1 h-1, and growth was saturated at approximately 0.45 d-1 for both MED and SARG strains at 18[deg]C, but saturating irradiances differed between strains. Atlantic strains exhibited increased light-saturated rates and quantum yield for carbon fixation under blue light.     

Effect of Anoxia on Carbohydrate Metabolism in Rice Seedlings

The metabolism of carbohydrates was investigated in rice (Oryza sativa L.) seedlings grown under anoxia. Two phases can be recognized in the utilization of carbohydrates: during the first days of germination under anoxia, the metabolism of sugars is mainly degradative, whereas after the induction of [alpha]-amylase (EC 3.2.1.1) has taken place, the increased presence of glucose and sucrose indicates that both starch degradation and sucrose synthesis operate. The analysis of the enzymes involved in carbohydrate metabolism indicates that anoxic rice seedlings possess a set of enzymes that allow the efficient metabolism of starch and sucrose to fructose-6-phosphate. We propose that cytosolic sucrose metabolism in anoxic rice seedlings takes place mainly through a sucrose synthase (EC 2.4.1.13) pathway with nucleoside diphosphate kinase (EC 2.7.4.6), allowing the cycling of urydilates needed for the operation of this pathway.     

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.     

Carbohydrate metabolism during postharvest ripening in kiwifruit

Mature fruit (kiwifruit) of Actinidia deliciosa var. deliciosa (A. Chev.), (C.F.) Liang and Ferguson cv. Haywood (Chinese gooseberry) were harvested and allowed to ripen in the dark at 20° C. Changes were recorded in metabolites, starch and sugars, adenine nucleotides, respiration, and sucrose and glycolytic enzymes during the initiation of starch degradation, net starch-to-sucrose conversion and the respiratory climacteric. The conversion of starch to sucrose was not accompanied by a consistent increase in hexose-phosphates, and UDP-glucose declined. The activity of sucrose phosphate synthase (SPS) measured with saturating substrate rose soon after harvesting and long before net sucrose synthesis commenced. The onset of sugar accumulation correlated with an increase in SPS activity measured with limiting substrates. Throughout ripening, until sucrose accumulation ceased, feeding [¹⁴C] glucose led to labelling of sucrose and fructose, providing evidence for a cycle of sucrose synthesis and degradation. It is suggested that activation of SPS, amplified by futile cycles, may regulate the conversion of starch to sugars. The respiratory climacteric was delayed, compared with net starchsugar interconversion, and was accompanied by a general decline of pyruvate and all the glycolytic intermediates except fructose-1,6-bisphosphate. The ATP/ ADP ratio was maintained or even increased. It is argued that the respiratory climacteric cannot be simply a consequence of increased availability of respiratory substrate during starch-sugar conversion, nor can it result from an increased demand for ATP during this process.Abbreviations Frul,6bisP fructose-1,6-bisphosphate - Frul,6Pase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - SPS sucrose phosphate synthase - UDPGlc uridine 5'-diphosphoglucose We thank Professor G. Costa, University of Udine and Flavia Succhi, University of Bologna for their help in obtaining the fruit in Italy. E.A.M. was the recipient of a travel grant through the NZ/German Technological Agreement.     

Transgenic potato plants with strongly decreased expression of pyrophosphate:fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers

Potato (Solanum tuberosum L.) plants were transformed with antisense constructs to the genes encoding the -and -subunits of pyrophosphate: fructose-6-phosphate phosphotransferase (PEP), their expression being driven by the constitutive CaMV 35S promotor. (i) In several independent transformant lines, PFP expression was decreased by 70–90% in growing tubers and by 88–99% in stored tubers. (ii) The plants did not show any visual phenotype, reduction of growth or decrease in total tuber yield. However, the tubers contained 20–40% less starch than the wild type. Sucrose levels were slightly increased in growing tubers, but not at other stages. The rates of accumulation of sucrose and free hexoses when tubers were stored at 4° C and the final amount accumulated were the same in antisense and wild-type tubers. (iii) Metabolites were investigated at four different stages in tuber life history; growing (sink) tubers, mature tubers, cold-sweetening tubers and sprouting (source) tubers. At all stages, compared to the wild type, antisense tubers contained slightly more hexose-phosphates, two- to threefold less glycerate-3-phosphate and phosphoenolpyruvate and up to four-to fivefold more fructose-2,6-bisphosphate. (iv) There was no accumulation or depletion of inorganic pyrophosphate (PPi), or of UDP-glucose relative to the hexose-phosphates. (v) The pyruvate content was unaltered or only marginally decreased, and the ATP/ADP ratio did not change. (vi) Labelling experiments on intact tubers did not reveal any significant decrease in the unidirectional rate of metabolism of [U-¹⁴C]sucrose to starch, organic acids or amino acids. Stored tubers with an extreme (90%) reduction of PFP showed a 25% decrease in the metabolism of [U¹⁴-C] sucrose. (vii) Metabolism (cycling) of [U-¹⁴C]glucose to surcrose increased 15-fold in discs from growing antisense tubers, compared with growing wild-type tubers. Resynthesis of sucrose was increased by 10–20% when discs from antisense and wild-type tubers stored at 4° C (cold sweetening) were compared. The conversion of [U-¹⁴C]glucose to starch was decreased by about 30% and 50%, respectively. (viii) The randomisation of [1-¹³C]glucose in the glucosyl and fructosyl moieties of sucrose was decreased from 13.8 and 15.7% in the wild type to 3.6 and 3.9% in an antisense transformant. Simultaneously, randomisation in glucosyl residues isolated from starch was reduced from 14.4 to 4.1%. (ix) These results provide evidence that PFP catalyses a readily reversible reaction in tubers, which is responsible for the recycling of label from triose-phosphates to hexose-phosphates, but with the net reaction in the glycolytic direction. The results do not support the notion that PFP is involved in regulating the cytosolic PPi concentration. They also demonstrate that PFP does not control the rate of glycolysis, and that tubers contain exessive capacity to phosphorylate fructose-6-phosphate. The decreased concentration of phosphoenolpyruvate and glycerate-3-phosphate compensates for the decrease of PFP protein by stimulating ATP-dependent phosphofructokinase, and by stimulating fructose-6-phosphate,2-kinase to increase the fructose-2,6-bisphosphate concentration and activate the residual PFP. The decreased starch accumulation is explained as an indirect effect, caused by the increased rate of resynthesis (cycling) of sucrose in the antisense tubers.Abbreviations Fru1,6bisP fructose-1,6-bisphosphate - Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose-6-phosphate - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - NMR nuclear magnetic resonance - 3PGA glycerate-3-phosphate - PEP phosphoenolpyruvate - PEP pyrophosphate: fructose-6-phosphate phosphotransferase - PFK phosphofructokinase - UDPGlc UDP glucose - WT wild type This research was supported by the Bundesministerium for Forschung and Technology (M.S., U.S.), the Canadian Research Council (S.C., D.D.), the Agricultural and Food Research Council (R.V.) and Sandoz Agro Ltd. (M.H., M.S.).     

Chloroplast Response in Dunaliella salina to Irradiance Stress (Effect on Thylakoid Membrane Protein Assembly and Function)

The chloroplast response in the green alga Dunaliella salina to irradiance stress was investigated. Cells were grown under low light (LL) at 100 [mu]mol photons m-2 s-1 or high light (HL) at 2000 [mu]mol photons m-2 s-1 incident intensity. LL-grown cells had a low chlorophyll (Chl) a/b ratio, an abundance of light-harvesting complex II proteins (LHC-II), and a large Chl antenna size. HL-grown cells had a higher Chl a/b ratio, relatively fewer LHC-II, and a small Chl antenna size. The more abundant higher molecular mass subunits of the LHC-II (approximately 31 kD) were selectively depleted from the thylakoid membrane of HL-grown cells. Light-shift experiments defined the kinetics of change in the subunit composition of the LHC-II and suggested distinct mechanisms in the acclimation of thylakoids to HL or LL conditions. The results showed that irradiance exerts a differential regulation on the expression of various Lhcb genes. The specific polyclonal antibodies used in this work, raised against the purified LHC-II, cross-reacted with a polypeptide of approximately 20 kD in HL-grown samples. In this work we examined the dynamics of induction of this novel protein and discuss its function in terms of a chloroplast response to the level of irradiance.     

Photosynthetic Characteristics of Segregates from Hybrids between Flaveria brownii (C4 Like) and Flaveria linearis (C3-C4)

Characteristics related to C4 photosynthesis were studied in reciprocal F1 hybrids and F2 plants from Flaveria brownii (C4 like) and Flaveria linearis (C3-C4). The reciprocal F1 plants differed in 13C/12C ratios of leaves and the percentage of 14C initially incorporated into C4 acids, being more like the pollen parents in these traits. They did not differ in apparent photosynthesis or in O2 inhibition of apparent photosynthesis and differed only slightly in CO2 compensation concentration at 175 [mu]mol quanta m-2 s-1 and 400 mL L-1 O2. The 13C/12C ratios of 78 F2 progeny from the two F1 plants exhibited a normal distribution centered between those of the parents, with a few values slightly higher and lower than the parents. Apparent photosynthesis at 130 [mu]L L-1 CO2 and inhibition of photosynthesis by O2 was nearly normally distributed in the F2 population, but no values for F2 plants approached those for F. brownii (15.4 [mu]mol m-2 s-1 and 7.8%, respectively). Distribution of the CO2 compensation concentration measured at 1000 [mu]mol quanta m-2 s-1 and 400 mL L-1 of O2 in the F2 population was skewed toward F. brownii with 72% of the progeny having values <9 [mu]L of CO2 L-1 compared to 1.5 and 27.2 [mu]L L-1 for F. brownii and F. linearis, respectively. Correlations among traits of F2 plants were low (coefficients of 0.30 to -0.49), indicating that the C4- related traits are not closely linked in segregating populations. Plants in the F2 population selected for high or low apparent photosynthesis at 130 [mu]L of CO2 L-1 (six each) did not rank consistently high or low for 13C/12C ratios, O2 inhibition of apparent photosynthesis, CO2 compensation concentration, or activities of phosphoenolpyruvate carboxylase or NADP-malic enzyme. This study confirms results of earlier work that indicates independent segregation of C4 traits and also shows that the C4-like parental type can be recovered, at least for some characteristics (13C/12C ratio), in segregating populations. Recovery of fully functional C4 plants awaits further experimentation with C4 x C3 or C4 x C3-C4 hybrid plants that produce fertile progeny.