Growth of cotton under continuous salinity stress: influence on allocation pattern,stomatal and non-stomatal components of photosynthesis and dissipation of excess light energy

Cotton (Gossypium hirsutum L.) plants were grown in flowing-culture solutions containing 0%, 26% and 55% natural seawater under controlled and otherwise identical conditions. Leaf Na⁺ content rose to 360 mM in 55% seawater, yet the K⁺ content was maintained above 100 mM. The K⁺/Na⁺ selectivity ratio was much greater in the saline plants than in the control plants. All plants were healthy and able to complete the life cycle but relative growth rate fell by 46% in 26% seawater and by 83% in 55% seawater. Much of this reduction in growth was caused by a decreased allocation of carbon to leaf growth versus root growth. The ratio of leaf area/plant dry weight fell by 32% in 26% seawater and by 50% in 55 % seawater while the rate of carbon gain per unit leaf area fell by only 20% in 26% seawater and by as much as 66% in 55% seawater. Partial stomatal closure accounted for nearly all of the fall in the photosynthesis rate in 26% seawater but in 55% seawater much of the fall also can be attributed to non-stomatal factors. As a result of the greater effect of salinity on stomatal conductance than on CO₂-uptake rate, photosynthetic water-use efficiency was markedly improved by salinity. This was also confirmed by stablecarbon-isotope analyses of leaf sugar and of leaf cellulose and starch. — Although non-stomatal photosynthetic capacity at the growth light was reduced by as much as 42% in 55% seawater, no effects were detected on the intrinsic photon yield of photosynthesis nor on the efficiency of photosystem II photochemistry, chlorophyll a/b ratio, carotenoid composition or the operation of the xanthophyll cycle. Whereas salinity caused in increase in mesophyll thickness and content of chloroplast pigments it caused a decrease in total leaf nitrogen content. The results indicate that the salinity-induced reduction in non-stomatal photosynthetic capacity was not caused by any detrimental effect on the photosynthetic apparatus but reflects a decreased allocation to enzymes of carbon fixation. — Rates of energy dissipation via CO₂ fixation and photorespiration, calculated from gas-exchange measurements, were insufficient to balance the rate of light-energy absorption at the growth light. Salinity therefore would have been expected to cause the excess excitation energy to rise, leading to an increased nonradiative dissipation in the pigment bed and resulting increases in non-photochemical fluorescence quenching and zeaxanthin formation. However, no such changes could be detected, implying that salinity may have increased energy dissipation via a yet unidentified energy-consuming process. This lack of a response to salinity stress is in contrast to the responses elicited by short-term water stress which caused strong non-photochemical quenching and massive zeaxanthin formation.Abbreviations and Symbols A net rate of CO₂ uptake - A_c calculated rate of CO₂ uptake at constant p_i - Chl chlorophyll - E rate of transpiration - EPS epoxidation state of xanthophyll cycle components - F, F_m fluorescence emission at the actual, full reduction of PSII reaction centers - F_v variable fluorescence - g_s stomatal conductance to water vapor - g_w conductance to CO₂transfer from intercellular spaces to chloroplasts - NPQ non-photochemical fluorescence quenching - p_a, p_i, p_c atmospheric, intercellular and chloroplastic partial pressures of CO₂ - PCRO photosynthetic carbon reduction and oxygenation cycle sum of the rate of carboxylation and oxygenation - PFD photon flux density - PSII photosystem II - V+A+Z pool size of xanthophyll cycle components - ¹³C carbon-isotope composition - (_PSII) photon yield of PSII photochemistry at the actual reduction state in the light * C.I.W.-D.P.B. Publication No. 1115, CNR-RAISA paper No. XXXWe thank Connie Shih for skilful assistance in growing plants and for conducting HPLC analyses and Barbara Mortimer for conducting the nitrogen analyses. Thanks are also due to C. Barry Osmond (now, Australian National University, Research School of Biological Sciences, Canberra) and Larry Giles of the Department of Botany, Duke University, Durham, N.C., for conducting carbonisotope analysis. E.B. was partially supported by the National Research Council of Italy, Special Project RAISA, Sub-Project No. 2. A Carnegie Institution Fellowship to E.B. is also gratefully acknowledged. This work was supported by grant No. 89-37-280-4902 of the Competitive Grant Program of the U.S. Department of Agriculture to O.B.     

Dynamics of the xanthophyll cycle and non-radiative dissipation of absorbed light energy during exposure of Norway spruce to high irradiance

The response of Norway spruce saplings (Picea abies [L.] Karst.) was monitored continuously during short-term exposure (10 days) to high irradiance (HI; 1000mumolm(-2)s(-1)). Compared with plants acclimated to low irradiance (100mumolm(-2)s(-1)), plants after HI exposure were characterized by a significantly reduced CO(2) assimilation rate throughout the light response curve. Pigment contents varied only slightly during HI exposure, but a rapid and strong response was observed in xanthophyll cycle activity, particularly within the first 3 days of the HI treatment. Both violaxanthin convertibility under HI and the amount of zeaxanthin pool sustained in darkness increased markedly under HI conditions. These changes were accompanied by an enhanced non-radiative dissipation of absorbed light energy (NRD) and the acceleration of induction of both NRD and de-epoxidation of the xanthophyll cycle pigments. We found a strong negative linear correlation between the amount of sustained de-epoxidized xanthophylls and the photosystem II (PSII) photochemical efficiency (F(V)/F(M)), indicating photoprotective down-regulation of the PSII function. Recovery of F(V)/F(M) at the end of the HI treatment revealed that Norway spruce was able to cope with a 10-fold elevated irradiance due particularly to an efficient NRD within the PSII antenna that was associated with enhanced violaxanthin convertibility and a light-induced accumulation of zeaxanthin that persisted in darkness.     

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

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 (A ₅₀₅) and in the contents of antheraxanthin and zeaxanthin that occur upon exposure of predarkened leaves to excessive light. A linear relationship between A 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.Abbreviations and Symbols Chl chlorophyll - DTT dithiothreitol - EPS epoxidation state - NPQ non-photochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - F, F_m fluorescence emission at the actual, full closure of the PSII centers C.I.W.-D.P.B. Publication No. 1092We thank Connie Shih for skillful assistance in growing the plants, for conducting the HPLC analyses, and for preparing the figures. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W.B. is gratefully acknowledged. This work was supported by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B.     

The relationship between phosphate status and photosynthesis in leaves

Spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.) were grown in hydroponic culture with varying levels of orthophosphate (Pi). When leaves were fed with 20 mmol·l^–1 Pi at low CO₂ concentrations, a temporary increase of CO₂ uptake was observed in Pi-deficient leaves but not in those from plants grown at 1 mmol·l^–1 Pi. At high concentrations of CO₂ (at 21% or 2% O₂) the Pi-induced stimulation of CO₂ uptake was pronounced in the Pi-deficient leaves. The contents of phosphorylated metabolites in the leaves decreased as a result of Pi deficiency but were restored by Pi feeding. These results demonstrate that there is an appreciable capacity for rapid Pi uptake by leaf mesophyll cells and show that the effects of long-term phosphate deficiency on photosynthesis may be reversed (at least temporarily) within minutes by feeding with Pi.Abbreviation Pi orthophosphate     

Adenine nucleotides and the xanthophyll cycle in leaves

The relationships among the leaf adenylate energy charge, the xanthophyll-cycle components, and photosystem II (PSII) fluorescence quenching were determined in leaves of cotton (Gossypium hirsutum L. cv. Acala) under different leaf temperatures and different intercellular CO₂ concentrations (C_i). Attenuating the rate of photosynthesis by lowering the C_i at a given temperature and photon flux density increased the concentration of high-energy adenylate phosphate bonds (adenylate energy charge) in the cell by restricting ATP consumption (A.M. Gilmore, O. Björkman 1994, Planta 192, 526–536). In this study we show that decreases in photosynthesis and increases in the adenylate energy charge at steady state were both correlated with decreases in PSII photo-chemical efficiency as determined by chlorophyll fluorescence analysis. Attenuating photosynthesis by decreasing C_i also stimulated violaxanthin-de-epoxidation-dependent nonradiative dissipation (NRD) of excess energy in PSII, measured by nonphotochemical fluorescence quenching. However, high NRD levels, which indicate a large trans-thylakoid proton gradient, were not dependent on a high adenylate energy charge, especially at low temperatures. Moreover, dithiothreitol at concentrations sufficient to fully inhibit violaxanthin de-epoxidation and strongly inhibit NRD, affected neither the increased adenylate energy charge nor the decreased PSII photo-chemical efficiency that result from inhibiting photosynthesis. The build-up of a high adenylate energy charge in the light that took place at low C_i and low temperatures was accompanied by a slowing of the relaxation of non-photochemical fluorescence quenching after darkening. This slowly relaxing component of nonphotochemical quenching was also correlated with a sustained high adenylate energy charge in the dark. These results indicate that hydrolysis of ATP that accumulated in the light may acidify the lumen and thus sustain the level of NRD for extended periods after darkening the leaf. Hence, sustained nonphotochemical quenching often observed in leaves subjected to stress, rather than being indicative of photoinhibitory damage, apparently reflects the continued operation of NRD, a photoprotective process.Abbreviations A antheraxanthin - adenylate kinase (myokinase), ATP:AMPphosphotransferase - C_i intercellular CO₂ concentration - DPS de-epoxidation state of violaxanthin, ([Z+A]/[V+A+Z]) - DTT dithiothreitol - pH trans-thylakoid proton gradient - [2ATP+ADP] - F steady-state fluorescence in the presence of NRD - F_M maximal fluorescence in the absence of NRD - F_M maximal fluorescence in the presence of NRD - NRD nonradiative energy dissipation - PET photosynthetic electron transport rate - PFD photon flux density - _PSII photon yield of PSII photochemistry at the actual reduction state in the light or dark - Q_A the primary electron acceptor of PSII - [ATP+ADP+AMP] - SV_N Stern-Volmer nonphotochemical quenching - V violaxanthin - Z zeaxanthin We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. A Carnegie Institution Fellowship to A.G. is also gratefully acknowledged.     

The intracellular distribution of adenylate kinase in the leaves of spinach,wheat and barley

Of the total adenylate-kinase activity in 10-d-old barley and wheat leaves, 40–50% is localised in the chloroplasts, while in mature spinach leaves 50–70% of the enzyme is chloroplastic. The extra-chloroplastic adenylate-kinase activity is associated with the mitochondria, very little, if any, is freely soluble in the cytoplasm. The adenylate pool of the cytoplasm could have access to adenylate-kinase activity in the intermitochondrial space because of the free permeation of adenylates across the outer mitochondrial membrane. Thus the adenylate pool of the cytoplasm could be subject to adenylate-kinase equilibrium. The mitochondrial adenylate kinase appeared to the localised exclusively in the intermembrane space.     

Metabolite levels in the stroma of spinach chloroplasts exposed to osmotic stress: Effects of the pH of the medium and exogenous dihydroxyacetone phosphate

The levels of stromal photosynthetic intermediates were measured in isolated intact spinach (Spinacia oleracea L.) chloroplasts exposed to reduced osmotic potentials. Stressed chloroplasts showed slower rates of metabolite accumulation upon illumination than controls. Relative to other metabolites sedoheptulose-1,7-bisphosphate (SBP) and fructose-1,6-bisphosphate (FBP) accumulated in the stroma in the stressed treatments. Under these conditions 3-phosphoglycerate (3-PGA) efflux to the medium was restricted. Chloroplasts previously incubated with [³²P]KH₂PO₄ and [³²P]dihydroxyacetone phosphate ([³²P]DAP) in the dark were characterized by very high FBP and SBP levels prior to illumination. Metabolism of these pools upon illumination increased with increasing pH of the medium but was consistently inhibited in osmotically stressed chloroplasts. The responses of stromal FBP and SBP pools under hypertonic conditions are discussed in terms of both inhibited light activation of fructose-1,6-bisphosphatase (EC 3.1.3.11) and sedoheptulose-1,7-bisphosphatase (EC 3.1.3.37), and likely increases in stromal ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) active-site concentrations.Abbreviations and symbols DAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate - SBP sedoheptulose-1,7-bisphosphate - _s osmotic potential     

The super-excess energy dissipation in diatom algae: comparative analysis with higher plants

When grown at intermittent light regime, diatom alga Phaeodactylum tricornutum is able to form photoprotective non-photochemical chlorophyll fluorescence quenching (NPQ) three to five times larger than that observed in the higher plants. This quenching is sustained in the dark for 5 to 10 min, reverses completely within approximately 1 h and seems to be very tightly related to the presence of the zeaxanthin analogue, diatoxanthin. Addition of the uncoupler NH4Cl before illumination can completely abolish formation of NPQ, revealing the ΔpH-dependency of the xanthophyll cycle activity. Once established, NPQ can also be almost completely reversed by the uncoupler. However, the higher NPQ is formed the more time is required for its reversal. At the point when the fluorescence was approximately 90% recovered the level of illumination-induced diatoxanthin was found to be only partially reduced. This indicates that the proton gradient is a key triggering factor of NPQ. It was also noticed that NPQ in Phaeodactylum cells was absent even when majority of reaction centers were closed and the plastoquinone pool was significantly reduced. The absence of NPQ at these conditions could be due to very low levels of ΔpH. It is likely that in diatoms alternative sources of protons such as the PS I cyclic electron transfer and/or chlororespiration are important in generating the proton gradient sufficient to trigger NPQ. Absorption changes associated with the xanthophyll cycle activity were found to be larger than those for higher plants. The position of the positive maximum in the difference spectrum illuminated-minus-dark was 512–514 nm in comparison to the 505–508 nm for leaves. The 535 nm band associated with NPQ in plants is absent in Phaeodactylum. An uncoupler-sensitive absorption change at 522 nm was discovered. Kinetics of NPQ showed linear correlation with the 522 nm absorption change. This revised version was published online in June 2006 with corrections to the Cover Date.     

Relationships among violaxanthin deepoxidation,thylakoid membrane conformation,and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (Z; peak at 506 nm) and by light scattering (S; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (S_g) for absorbance changes around 540 nm that are due to a spectral overlap with Z. This protocol isolated the component which is caused by light-scattering changes alone (S_n). In control leaves, the kinetics and temperature dependence of the initial rate of rise in S_n that takes place upon illumination, closely matched that of Z. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both S_n and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on A_n and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of S_n and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of Z upon darkening was much slower than the relaxation of S_n and NPQ. The relaxation of S_n and NPQ showed quantitatively similar kinetics and temperature dependencies (Q₁₀=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause S_n or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by S_n. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained S_n and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.Abbreviations A antheraxanthin - DTT dithiothreitol - F, F_m chlorophyll fluorescence yield at actual, full closure of the PSII centers - NPQ nonphotochemical chlorophyll fluorescence quenching - PFD photon flux density - PSII photosystem II - V violaxanthin - Z zeaxanthin - S_n, Z spectral absorbance change caused by light-scattering, violaxanthin deepoxidation We thank Connie Shih for skillful assistance in growing the plants, and for conducting HPLC analyses. A Carnegie Institution Fellowship and a Feodor-Lynen-Fellowship by the Alexander von Humboldt-Foundation to W. B. is gratefully acknowledged. This work was supported in part by Grant No. 89-37-280-4902 of the Competitive Grants Program of the U.S. Department of Agriculture to O.B. This is C. I. W. — D. P. B. Publication No. 1094.     

Inhibition of photosynthesis,acidification and stimulation of zeaxanthin formation in leaves by sulfur dioxide and reversal of these effects

Leaves of Pelargonium zonale L. and Spinacia oleracea L. were fumigated with high concentrations of SO₂ for very short periods of time with the aim of first producing acute symptoms of damage and then observing repair. The response of different photosynthetic parameters to SO₂ was monitored during and after fumigation. The following results were obtained: (1) Inhibition of CO₂ assimilation in the light was accompanied by increased reduction of the quinone acceptor, Q_A, of photosystem II and by increased oxidation of the electrondonor pigment P₇₀₀ of photosystem I. Increased control of photosystem II activity in the SO₂-inhibited state was also indicated by increased light scattering and by increased non-photochemical quenching of chlorophyll fluorescence. Both are indicators of chloroplast energization. Apparently, SO₂ did not decrease but rather increased energization of the chloroplast thylakoid system by light. (2) Accumulation of dihydroxyacetone phosphate, fructose-1,6-phosphate and ribulose-1,5-phosphate and a decrease of 3-phosphoglycerate and hexosephosphate indicated that SO₂ inhibited enzymes of the Calvin cycle. (3) Stimulated postillumination CO₂ evolution suggested that when photosynthesis declined respiration increased to provide energy for repair reactions. (4) Increased leaf absorbance at 505 nm indicated increased stimulation of zeaxanthin formation in thylakoid membranes under the influence of SO₂. A similar increase in 505-nm absorbance could be induced by high concentrations of CO₂. In darkened leaves, SO₂ did not produce changes in 505-nm absorbance. (5) While zeaxanthin formation was stimulated, changes in the fluorescence of the pH-indicating dye pyranine, which had been fed to the leaves, indicated acidification of the cytoplasm of leaf cells by SO₂. Maximum acid production by SO₂ required light. In contrast, cytoplasmic acidification of leaf cells by CO₂ was similar in the light and in the dark. (6) Since zeaxanthin formation is known to depend on the acidification of the thylakoid lumen, SO₂-dependent zeaxanthin formation indicated SO₂-dependent acidification of the thylakoid lumen as the indirect result of cytoplasmic acidification by SO₂. (7) Inhibition of photosynthesis and other effects of SO₂ were fully reversible in the light. Detoxification of SO₂ and reactivation of the photosynthetic apparatus were slow or absent in the dark. Light had a dual effect on the action of SO₂. Transiently, it first increased the extent of inhibition of assimilation, but, finally, it reversed inhibition. Sulfur dioxide was inhibitory as a consequence of the chemical reactivity of its hydration products rather than as a result of cellular acidification by the produced acid. The initial acidification was followed by an appreciable alkalisation demonstrating the action of the pH-stat mechanism. (8) The data are discussed in relation to SO₂ toxicity under field conditions when plants are chronically exposed to polluted air.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - F6P fructoce-6-phosphate - F, Fm, Fm, Fo, Fo chlorophyll fluorescence levels - PGA 3-phosphoglycerate - P₇₀₀ primary donor of photosystem I - Q_A primary quinone acceptor of photosystem II - q_p photochemical quenching of chlorophyll fluorescence - NPQ non-photochemical quenching of chlorophyll fluorescence - RuBP ribulose-1,5-bisphosphate Dedicated to Professor O.L. Lange on the occasion of his 65th birthdayOn leave from the Centre for Multidisciplinary Sciences, University of Belgrade, YugoslaviaThis work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 251 of the University of Würzburg. S. V.-J. acknowledges support by the Leibniz program of the Deutsche Forschungsgemeinschaft and by the Fonds for Science of the Republic of Serbia (contract no. 8604). We are grateful to Drs. Z.-H. Yin, U. Takahama and K.-J. Dietz (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, FRG) for cooperation and helpful discussions.     

Photoinhibition of photosynthesis under natural conditions in ivy (Hedera helix L.) growing in an understory of deciduous trees

We investigated to what extent south-exposed leaves (E-leaves) of the evergreen ivy (Hedera helix L.) growing in the shadow of two deciduous trees suffered from photoinhibition of photosynthesis when leaf-shedding started in autumn. Since air temperatures drop concomitantly with increase in light levels, changes in photosynthetic parameters (apparent quantum yield, _i and maximal photosynthetic capacity of O₂ evolution, P_max; chlorophyll-a fluorescence at room temperature) as well as pigment composition were compared with those in north-exposed leaves of the same clone (N-leaves; photosynthetic photon flux density PPFD< 100 mol · m^–2 · s^–2) and phenotypic sun leaves (S-leaves; PPFD up to 2000 mol · m^–2 · s^–1).In leaves exposed to drastic light changes during winter (E-leaves) strong photoinhibition of photosynthesis could be observed as soon as the incident PPFD increased in autumn. In contrast, in N-leaves the ratio of variable fluorescence to maximum fluorescence (F_V/F_Mm) and _i did not decline appreciably prior to severe frosts (up to -12° C) in January. At this time, _i was reduced to a similar extent in all leaves, from about 0.073 mol O₂ · mol^–1 photons before stress to about 0.020. Changes in _i were linearly correlated with changes in f_v/f_m (r = 0.955). The strong reduction in F_V/F_M on exposure to stress was caused by quenching in F_M. The initial fluorescence (F₀), however, was also quenched in all leaves. The diminished fluorescence yield was accompanied by an increase in zeaxanthin content. These effects indicate that winter stress in ivy primarily induces an increase in non-radiative energy-dissipation followed by photoinhibitory damage of PSII. Although a pronounced photooxidative bleaching of chloroplast pigments occurred in January (especially in E-leaves), photosynthetic parameters recovered completely in spring. Thus, the reduction in potential photosynthetic yield in winter may be up to three times greater in leaves subjected to increasing light levels than in leaves not exposed to a changing light environment.Abbreviations and Symbols F₀, F_M initial and maximal fluorescence yield when all PSII centres are open and closed - F_V variable fluorescence (F_M-F₀) - P_max maximal photosynthetic capacity at 1000 umol · m^–2 · s^–1 PPFD and CO₂ saturation - PPFD photosynthetic photon flux density - _i apparent quantum yield of photosynthetic O₂ evolution - E-leaves, N-leaves shade leaves exposed, not exposed to drastic light changes during winter - S-leaves sun leaves from an open ivy stand Dedicated to Professor Otto Härtel on the occasion of his 80th birthdayThis work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

Correlation between persistent forms of zeaxanthin-dependent energy dissipation and thylakoid protein phosphorylation

High light stress induced not only a sustained form of xanthophyll cycle-dependent energy dissipation but also sustained thylakoid protein phosphorylation. The effect of protein phosphatase inhibitors (fluoride and molybdate ions) on recovery from a 1-h exposure to a high PFD was examined in leaf discs of Parthenocissus quinquefolia (Virginia creeper). Inhibition of protein dephosphorylation induced zeaxanthin retention and sustained energy dissipation (NPQ) upon return to low PFD for recovery, but had no significant effects on pigment and Chl fluorescence characteristics under high light exposure. In addition, whole plants of Monstera deliciosa and spinach grown at low to moderate PFDs were transferred to high PFDs, and thylakoid protein phosphorylation pattern (assessed with anti-phosphothreonine antibody) as well as pigment and Chl fluorescence characteristics were examined over several days. A correlation was obtained between dark-sustained D1/D2 phosphorylation and dark-sustained zeaxanthin retention and maintenance of PS II in a state primed for energy dissipation in both species. The degree of these dark-sustained phenomena was more pronounced in M. deliciosa compared with spinach. Moreover, M. deliciosa but not spinach plants showed unusual phosphorylation patterns of Lhcb proteins with pronounced dark-sustained Lhcb phosphorylation even under low PFD growth conditions. Subsequent to the transfer to a high PFD, dark-sustained Lhcb protein phosphorylation was further enhanced. Thus, phosphorylation patterns of D1/D2 and Lhcb proteins differed from each other as well as among plant species. The results presented here suggest an association between dark-sustained D1/D2 phosphorylation and sustained retention of zeaxanthin and energy dissipation (NPQ) in light-stressed, and particularly photoinhibited, leaves. Functional implications of these observations are discussed.This revised version was published online in October 2005 with corrections to the Cover Date.     

Light stimulation of proline synthesis in water-stressed barley leaves

The effect of light on [¹⁴C]glutamate conversion to free proline during water stress was studied in attached barley (Hordeum vulgare L.) leaves which had been trimmed to 10 cm in length. Plants at the three-leaf stage were stressed by flooding the rooting medium with polyethylene glycol 6000 (osmotic potential-19 bars) for up to 3 d. During this time the free proline content of 10-cm second leaves rose from about 0.02 to 2 mol/leaf while free glutamate content remained steady at about 0.6 mol/leaf. In stressed leaves, the amount of [¹⁴C]glutamate converted to proline in a 3-h period of light or darkness was taken to reflect the in-vivo rate of proline biosynthesis because the following conditions were met: (a) free-glutamate levels were not significantly different in light and darkness; (b) both tracer [¹⁴C]-glutamate and [¹⁴C]proline were rapidly absorbed; (c) rates of [¹⁴C]proline oxidation and incorporation into protein were very slow. As leaf water potential fell, more [¹⁴C]glutamate was converted to proline in both light and darkness, but at any given water potential in the range-12 to-20 bars, illuminated leaves converted twice as much [¹⁴C]glutamate to proline.     

Remote sensing of the xanthophyll cycle and chlorophyll fluorescence in sunflower leaves and canopies

Summary Sudden illumination of sunflower (Helianthus annuus L. cv. CGL 208) leaves and canopies led to excess absorbed PFD and induced apparent reflectance changes in the green, red and near-infrared detectable with a remote spectroradiometer. The green shift, centered near 531 nm, was caused by reflectance changes associated with the de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin and with the chloroplast thylakoid pH gradient. The red (685 nm) and near-infrared (738 nm) signals were due to quenching of chlorophyll fluorescence. Remote sensing of shifts in these spectral regions provides non-destructive information on in situ photosynthetic performance and could lead to improved techniques for remote sensing of canopy photosynthesis.CIW Publication #1072     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Extracellular ice and cell shape in frost-stressed cereal leaves: A low-temperature scanning-electron-microscopy study

Low-temperature scanning electron microscopy was used to examine transverse fracture faces through cereal leaf pieces subjected to frost. Specimens were studied before and after sublimation of the ice. The position of extracellular ice in the leaf was inferred from the difference between the specimen before and after sublimation and from ridges and points which occurred in the extracellular ice during sublimation. Steps in the fracture surface indicated that the fracture plane passed through the extracellular ice crystals as well as through cells and also helped identify extracellular ice. The cells in controls were turgid and extracellular ice was absent. Leaf pieces from hardened rye were excised and frost-stressed to-3.3°,-21° and-72°C, cooling at 2–12°·h^-1. Cell collapse and extracellular ice were evident at-3.3°C and increased considerably by-21° C. At-21° and-72°C the leaf pieces were mainly filled with extracellular ice and there were few remaining gas spaces. The epidermal and mesophyll cells were laterally flattened, perpendicular to their attachment to adjacent cells, and phloem and vascular sheath cells were more irregularly deformed. Leaf pieces from tender barley were cooled at 2°C·min^-1 to-20° C; they were then mainly filled with extracellular ice, and the cells were highly collapsed as in the rye. In rye leaves frozen to-3.6° C before excision, ice crystals occurred in peri-vascular, sub-epidermal and intervening mesophyll spaces. In rye leaf pieces frozen to-3.3° C after excision or to-3.6° C before excision, mesophyll cells were partly collapsed even when not covered by ice, indicating that collapse of the cell wall, as well as the enclosed protoplast, was driven by dehydration. No gas or ice-filled spaces were found between wall and the enclosed protoplast. It is suggested that this can be explained without invoking chemical bonding between cell wall and plasma membrane: when the wall pores are filled by water, the pore size would reduce vapour pressure so making penetration of the wall by ice or gas less likely.Abbreviations SEM scanning electron microscopy     

The photosynthetic induction response in wheat leaves: net CO2 uptake,enzyme activation,and leaf metabolites

The photosynthetic induction response was studied in whole leaves of wheat (Triticum aestivum L.) following 5-min, 30-min and 10-h dark periods. After the 5-min dark treatment there was a rapid burst in the rate of photosynthesis upon illumination (half of maximum after 30s), followed by a slight decrease after 1.5 more min and then a gradual rise to the maximum rate. During this initial burst in photosynthesis, there was a rapid rise in the level of 3-phosphoglycerate (PGA) and a high PGA/triose-phosphate (triose-P) ratio was obtained. In addition, after the 5-min dark treatment, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39), ribulose-5-phosphate kinase (EC 2.7.1.19) and chloroplastic fructose-1,6-bisphosphatase (EC 3.1.3.11) maintained a relatively high state of activation, and maximum activation occurred within 1 min of illumination. The results indicate there is a high capacity for CO₂ fixation in the cycle upon illumination but attaining maximum rates requires an increase in the ribulose-1,5-bisphosphate (RuBP) pool (adjustment in triose-P utilization for carbohydrate synthesis versus RuBP synthesis). With both the 30-min and 10-h dark pretreatments there was only a slight rise in photosynthesis upon illumination, followed by a lag, then a gradual increase to steady-state (half-maximum rate after 6 min). In contrast to the 5-min dark treatment, the level of PGA was low and actually decreased initially, whereas the level of RuBP increased and was high during induction, indicating that Rubisco is limiting. This regulation via the carboxylase was not reflected in the initial extractable activity, which reached a maximum by 1 min after illumination. The light activation of chloroplastic fructose-1,6-bisphosphatase in leaves darkened for 30 min and 10 h prior to illumination was relatively slow (reaching a maximum after 8 min). However, this was not considered to limit carbon flux through the carbon-fixation cycle during induction since RuBP was not limiting. When photosynthesis approached the maximum steady-state rate, a high PGA/triose-P ratio and a high PGA/RuBP ratio were obtained. This may allow a high rate of photosynthesis by producing a favorable mass-action ratio for the reductive phase (the conversion of PGA to triose phosphate) while stimulating starch and sucrose synthesis.Abbreviations Chl chlorophyll - FBP fructose-1,6-bisphosphate - FBPase fructose-1,6-bisphosphatase - Fru6P fructose-6-phosphate - Glc6P glucose-6-phosphate - PGA 3-phosphoglycerate - Pi inoganic phosphate - Rubisco RuBP carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - Ru5P ribulose-5-phosphate - triose-P triose phosphates (dihydroxyacetone phosphate+glyceraldehyde-3-phosphate)     

The prime plasmalemma ATPase of the halophilic alga Dunaliella bioculata: purification and characterization

The prime plasmalemma ATPase of the halophilic green alga Dunaliella bioculata has been solubilized by Triton X-100 from a plasmalemma-rich membrane fraction and purified by anion-exchange chromatography. Vanadate-sensitive ATPase activity was totally enriched about 230-fold to a specific activity of approx. 250 nkat·mg protein^–1. The presence of Mg²⁺ or Mn²⁺ is essential for ATP hydrolysis by the enzyme. In addition to an equimolar requirement (11 Mg²⁺: ATP), there is further stimulation by Mg²⁺ (up to 20 mM) and by (100 mM) monovalent cations (K⁺ NH ₄ ⁺ >Rb⁺ -Na⁺ >Cs⁺ >Li⁺-choline⁺). Most anions have no or little effect. With a molecular mass of about 105 kDa for the single subunit, sensitivity to vanadate and N,N-dicyclohexylcarbodiimide (50% inhibition at about 1 M and 0.3 mM, respectively), strict ATP-specificity, and an acidic pH optimum, this enzyme shows the typical characteristics of the common type of H⁺-ATPase in the plasmalemma of higher plants and fungi. These results undermine the hypothesis of a wider distribution of a special (high salt) type of plasmalemma ATPase as found in the marine alga Acetabularia.Abbreviations BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - Mega-9 nonanoyl-N-methyl-glucamide - Mes N-morpholinoethanesulfonic acid - Mops N-morpholinopropanesulfonic acid - PAGE polyacrylamide-gel electrophoresis - PM plasmalemma-enriched membrane fraction - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft; we thank Drs. M. Ikeda and D. Oesterhelt (MPI für Biochemie, Martinsried, FRG) for generous and valuable information about their work prior to publication.     

Photosynthetic strategies in leaves and stems of Egeria densa

Photosynthetic mechanisms have been compared in leaves and, separately, in stems of Egeria densa Planch. In order to correlate the structural and functional characteristics of the two organs (1) the ultrastructural features of leaves and stems have been studied and (2) their photosynthetic activity has been evaluated by measuring in vivo both oxygen evolution and the kinetics of chlorophyll fluorescence. The results confirm the aquatic behaviour of the leaf which is able to utilize inorganic C supplied both as CO₂ and HCO ₃ ^– . In this respect, the different wall organization found in the two cell layers of the leaf is particularly interesting, since it could be related to the known polar mechanism of inorganic-C uptake. The stem, by contrast, behaves rather as an aerial organ, needing very high CO₂ concentrations in the aquatic environment in order to carry out photosynthesis. In the stem, the aerenchyma plays a role in supplying the green cells with gaseous respiratory CO₂, thus facilitating the photosynthetic activity of the submerged stems.The authors are grateful to C.U.G.A.S. (University of Padua) for the use of the scanning electron microscope. They also wish to thank Mr. Claudio Furlan and Mr. Giorgio Varotto for helpful technical assistance. This work was supported by a grant from C.N.R. and M.P.I, and was developed within the cooperation agreement between the Universities of Padova (Italy) and Innsbruck (Austria).