Posters Part 1

Schima superba and Pinus massoniana distributed over large areas in southern China both are dominant species at Dinghushan Biosphere Reserve. In the present study, the changes of chlorophyll fluorescence and xanthophyll cycle in the leaves of S. superba and P. massoniana exposed to simulated acid rain (SAR) were measured. When exposed to high light, the PSII photochemistry efficiency (F _v/F _m), efficiency of energy conversion in PSII (ΦPSII) and photochemical quenching (qP) of both S. superba and P. massoniana all decreased when acidity of SAR increased. Regarding non-photochemical quenching (qN), S. superba exposed to SAR had higher value than control plants, but there was no significant difference between the respective seedlings of P. massoniana. As for xanthophyll cycle of the two plant species, the leaves of S. superba exposed to SAR showed a higher content of carotenoids and a higher ability to convert violaxanthin to zeaxanthin than leaves of P. massoniana, which was consistent with S. superba exhibiting a stronger resistance to high light than P. massoniana. Although both species were susceptible to acid rain as shown by our results, P. massoniana was more susceptible compared to S. superba. These results provide an insight into how to protect the forest ecosystem at Dinghushan Biosphere Reserve.     

Dicyclohexylcarbodiimide alters the pH dependence of violaxanthin de-epoxidation

The de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle of higher plants is controlled by the pH of the thylakoid lumen. The influence of N,N′-dicyclohexylcarbodiimide (DCCD) on the pH dependence of the de-epoxidation reactions has been investigated in isolated pea thylakoids. In the presence of DCCD, the decrease in de-epoxidase activity at increasing pH was found to be shifted by about 0.3 pH units to more-alkaline pH values. This was paralleled by a less-pronounced cooperativity for the pH dependence of de-epoxidation. Comparative studies with antenna-depleted thylakoids from plants grown in intermittent light and with unstacked thylakoids indicated that binding of DCCD to antenna proteins is most probably not responsible for the altered pH dependence. Analyses of the zeaxanthin content of different antenna subcomplexes showed that the DCCD-induced de-epoxidation at high pH leads to zeaxanthin formation in all antenna proteins from both photosystems. Our data support the view that DCCD binding to the violaxanthin de-epoxidase may be responsible for the altered pH dependence. Received: 4 July 1998 / Accepted: 9 September 1998     

枸杞紫黄质脱环氧化酶(LcVDE)基因的克隆和分析

目的:克隆枸杞VDE基因的全长cDNA,通过对基因序列的生物信息学分析预测表达产物的结构特征和功能位点并验证其功能,为研究枸杞紫黄质循环的作用机理打下基础。方法:利用cDNA末端快速扩增和RT-PCR方法克隆枸杞VDE基因全长cDNA序列,生物软件分析VDE的生物学信息。构建VDE基因的原核表达载体pET-VDE,转化大肠后用IPTG诱导VDE过量表达;并构建体外反应体系对VDE表达蛋白酶功能进行验证。结果:LcVDE基因的ORF长1 413bp,编码的蛋白由470个氨基酸组成,分子量为53.61kDa,等电点为5.77。SDS-PAGE电泳结果表明,枸杞VDE基因在大肠杆菌中得到了过量表达。克隆基因表达蛋白进行紫黄质的脱环氧化反应,吸收光谱和HPLC的分析结果表明,表达蛋白催化了紫黄质的脱环氧化反应。结论:克隆得到的VDE基因编码的蛋白具有紫黄质脱环氧化酶的的功能与活性。     

The role of cis-carotenoids in abscisic acid biosynthesis

Evidence has been obtained which is consistent with 9-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography R_ts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15–20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D₂O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PA phaseic acid - t trans - Xan xanthoxin - flc flacca - not notabilis - sit sitiens The authors would like to thank the following for their help and advice: G. Britton (Department of Biochemistry, University of Liverpool, UK), B.H. Davies (Department of Biochemistry, University of Wales, Aberystwyth), P. Molnar, J. Szabolcs, D.C. Walton (Department of Biology, Suny, Syracuse, N.Y., USA), and Mr. J.K. Heald for his expert operation of the mass spectrometer. A.D.P. was supported initially by a Science and Engineering Research Council CASE award with Shell Biosciences, Sittingbourne, Kent, UK, and later by a Agricultural and Food Research Council (AFRC) grant. M.J.B. received a NATO fellowship. The mass spectrometer and HPLC-photodiode-array detector were purchased with funds provided by the AFRC.     

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 importance of a highly active and DeltapH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae

The present study focuses on the regulation of diatoxanthin (Dtx) epoxidation in the diadinoxanthin (Ddx) cycle containing algae Phaeodactylum tricornutum, Thalassiosira pseudonana, Cyclotella meneghiniana and Prymnesium parvum and its significance for the control of the photosystem II (PS II) antenna function. Our data show that Dtx epoxidase can exhibit extremely high activities when algal cells are transferred from high light (HL) to low light (LL). Under HL conditions, Dtx epoxidation is strongly inhibited by the light-driven proton gradient. Uncoupling of the cells during HL illumination restores the high epoxidation rates observed during LL. In Ddx cycle containing algae, non-photochemical quenching of chlorophyll fluorescence (NPQ) is directly correlated with the Dtx concentration and independent of the presence of the proton gradient. This means that a fast conversion of PS II from the heat dissipating state back to the light-harvesting state can only be realized by an efficient removal of the quenching pigment Dtx. It is proposed that the high Dtx epoxidation rates during LL illumination serve exactly this purpose. The inhibition of Dtx epoxidation by the DeltapH, on the other hand, ensures rapid increases in the Dtx concentration when photoprotection under conditions of HL illumination is required. The regulation of the PS II antenna function in Ddx cycle containing algae is different to that in violaxanthin (Vx) cycle containing plants, where for the zeaxanthin (Zx)-dependent NPQ the presence of a proton gradient is mandatory. In the green alga Chlorella vulgaris conversion of PS II from the heat dissipating state back to the light-harvesting state is controlled by the DeltapH, whose relaxation after a transition from HL to darkness or LL rapidly abolishes the thermal dissipation of excitation energy, including the Zx-dependent NPQ. Due to the inability of Zx to quench fluorescence in the absence of the DeltapH a fast epoxidation of Zx to Vx in LL is not needed and is missing in Chlorella vulgaris.     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis

Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.     

Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of trans-thylakoid ΔpH

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide-free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (ΔpH), epoxidation can proceed irrespective of a ΔpH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light-induced thylakoid ΔpH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid ΔpH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.