Suppression of zeaxanthin formation does not reduce photosynthesis and growth of transgenic tobacco under field conditions

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with an antisense cDNA construct of violaxanthin de-epoxidase (VDE) was examined for the effects of suppressed xanthophyll-cycle activity on photoinhibition, photosynthesis and growth under field conditions. De-epoxidation of violaxanthin and non-photochemical quenching were highly inhibited in antisense plants relative to vector-control and wild-type plants. However, no differences were observed between antisense and control plants in photosynthetic CO₂ uptake and maximum photochemical yield [(F_m–F_o)/F_m] measured at predawn or in actual photochemical yield [(F_m–F_s)/F_m] measured at midday. Moreover, growth rates of the plants were the same, as were the leaf area ratio, plant height and leaf number. Similarly, antisense plants did not exhibit greater susceptibility to photoinhibition than controls under field conditions. In contrast, when chloroplast protein (D1) synthesis was inhibited by lincomycin, antisense plants were more vulnerable to photoinhibition than wild-type plants. These results indicate that photoprotection under field conditions is not strictly dependent on the levels of the de-epoxidized xanthophylls, antheraxanthin and zeaxanthin.This revised version was published online in October 2005 with corrections to the Cover Date.     

Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration

The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a K_m for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single K_m±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.     

Expression of vde Gene Integrated Into Tobacco Genome in Antisense and Overexpressed Ways

Violaxanthin de-epoxidase (VDE) is localised in the thylakoid lumen of chloroplasts and catalyses de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin. Tobacco vde gene was inserted into a binary vector pCAMBIA1301 with the hygromycin resistant gene for selection in antisense and overexpressed ways. Two constructs with antisense and overexpressed vde gene were introduced in tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens strain LBA4404, PCR and Southern blot analyses demonstrated that the exogenous gene was integrated into genome of tobacco plants. VDE activity assay and HPLC analysis of pigments showed that the vde gene was expressed in the overexpressed transformants, whereas suppressed in the antisense ones. The chlorophyll fluorescence measurements proved that the contents of VDE in transgenic plants have a significant function in non-photochemical quenching.     

Transgenic tobacco with suppressed zeaxanthin formation is susceptible to stress-induced photoinhibition

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with the antisense construct of tobacco violaxanthin de-epoxidase was analyzed for responses in growth chambers to both short and long-term stress treatments. Following a short-term (2 or 3 h) high-light treatment, antisense plants had a greater reduction in F_v/F_m relative to wild-type, indicating a greater susceptibility to photoinhibition. The responses of antisense plants to long-term stress were examined in two separate experiments, one with high light alone and the other wherein high light and water stress were combined. In the light-stress experiment, plants were grown at 1300 mol photons m^–2 s^–1 under a 12 h photoperiod. In the light and water-stress experiment, plants were grown under moderately high light of 900 mol photons m^–2 s^–1, under a 16 h photoperiod, in combination with water stress. Both conditions caused formation of high antheraxanthin and zeaxanthin levels in wild-type plants but not in antisense plants. In both cases, antisense plants showed significant reductions in F_v/F_m and total leaf-pigment content relative to wild-type. The data demonstrate a critical photoprotective function of the xanthophyll cycle-dependent energy dissipation in tobacco exposed suddenly to high amounts of excess light over extended times.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

植物叶黄素循环的组成、功能和调节(综述)

对近几年来植物体内叶黄素循环的组成、功能、堇菜黄素脱环氧化酶和玉米黄素环氧化酶的结构、生化性质和调节,以及叶黄素的可转变性、定位等方面的研究进展作了综述。     

叶黄素循环组分的分离鉴定及紫黄质的大量制备

以薄层层析法(TLC)分离鉴定植物组织中叶黄素循环组分,建立了大量制备紫黄质的方法。此法所需时间短,分离效果好,检出灵敏度高,既适于少量样品的分离,也适于大样品的提纯制备,因此适合用于紫黄质的提取及大量制备。     

Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum

Based on our recent findings that in the diatom Phaeodactylum tricornutum, chlororespiration in periods of prolonged darkness leads to the accumulation of diatoxanthin (DT), we have elaborated in detail the interdependence between the chlororespiratory proton gradient and the activation of diadinoxanthin de-epoxidase (DDE). The data clearly demonstrates that activation of DDE in Phaeodactylum occurs at higher pH-values compared to activation of violaxanthin de-epoxidase (VDE) in higher plants. In thylakoid membranes as well as in enzyme assays with isolated DDE, the de-epoxidation of diadinoxanthin (DD) is efficiently catalyzed at pH 7.2. In comparison, de-epoxidation of violaxanthin (Vx) in spinach thylakoids is observed below pH 6.5. Phaeodactylum thylakoids isolated from high light grown cells, that also contain the pigments of the violaxanthin cycle, show violaxanthin de-epoxidation at higher pH-values, thus suggesting that in Phaeodactylum, one de-epoxidase converts both diadinoxanthin and violaxanthin. We conclude that the activation of DDE at higher pH-values can explain how the low rates of chlororespiratory electron flow, that lead to the build-up of a rather small proton gradient, can induce the observed accumulation of diatoxanthin in the dark. Furthermore, we show that dark activation of diadinoxanthin de-epoxidation is not restricted to Phaeodactylum tricornutum but was also found in another diatom, Cyclotella meneghiana     

植物叶片和冠层光化学反射指数与叶黄素循环的关系

以一串红(Salvia splendens Ker-Gawl.)和白车轴草(Trifolium repens Linn.)为材料,使用光谱反射技术测定了这两种植物叶片水平和冠层水平的光化学反射指数(PRI)的日变化,同时使用高效液相色谱分析法测定了这两种植物叶黄素循环的日变化,分析了单叶、群体冠层的PRI与叶黄素循环和叶片实际光化学效率(ΦPSⅡ)和非光化学淬灭(NPQ)之间的关系.结果表明,不论是叶片水平还是冠层水平,两种植物PRI的变化均与叶黄素的脱环氧化程度和NPQ之间呈显著的负相关、与ΦPSⅡ呈显著的正相关.研究结果表明无论叶片水平还是冠层水平上的光谱反射指数均能非常好地反映植物光合机构对光能的利用效率.     

Response of Tradescantia albiflora to growth irradiance: Change versus changeability

Most chloroplasts undergo changes in composition, function and structure in response to growth irradiance. However, Tradescantia albiflora, a facultative shade plant, is unable to modulate its light-harvesting components and has the same Chl a/Chl b ratios and number of functional PS II and PS I reaction centres on a Chl basis at all growth irradiances. With increasing growth irradiance, Tradescantia leaves have the same relative amount of chlorophyll—proteins of PS II and PS I, but increased xanthophyll cycle components and more zeaxanthin formation under high light. Despite high-light leaves having enhanced xanthophyll cycle content, all Tradescantia leaves acclimated to varying growth irradiances have similar non-photochemical quenching. These data strongly suggest that not all of the zeaxanthin formed under high light is necessarily non-covalently bound to major and minor light-harvesting proteins of both photosystems, but free zeaxanthin may be associated with LHC II and LHC I or located in the lipid bilayer. Under the unusual circumstances in light-acclimated Tradescantia where the numbers of functional PS II and PS I reaction centres and their antenna size are unaltered during growth under different irradiances, the extents of PS II photoinactivation by high irradiances are comparable. This is due to the extent of PS II photoinactivation being a light dosage effect that depends on the input (photon exposure, antenna size) and output (photosynthetic capacity, non-radiative dissipation) parameters, which in Tradescantia are not greatly varied by changes in growth irradiance.This revised version was published online in October 2005 with corrections to the Cover Date.     

缺锰降低大豆叶片叶绿素荧光的高能态猝灭

缺锰大豆叶片光合速率比对照低 5 0 %左右。缺锰叶片的Fv/Fm较对照低约 30 %～ 40 % ,而Fo比对照高 2～ 3倍左右。强光下 ,缺锰叶片的qP为对照的5 0 %。缺锰降低了叶片的高能态猝灭 (qE) ,仅占其NPQ的 6 0 %左右 ;而对照 qE组分占NPQ的 90 %左右。对照和缺锰叶片叶黄素库的脱环氧化程度分别为36 %和 2 1%左右。认为缺锰叶片 qE的降低可能是由于叶黄素脱环氧化程度减少所致     

The dissipation of excess excitation energy in British plant species

The reversible dissipation of excitation energy in higher plants is believed to protect against light-induced damage to the photosynthetic apparatus. This dissipation is measured as the non-photochemical quenching of chlorophyll fluorescence. A method is described whereby the saturated capacity for rapidly reversible non-photochemical quenching can be compared between plant species. This method was applied to 22 common British plant species whose habitat was quantified using an index that describes shade tolerance. An association was found between occurrence in open habitats and a high capacity for non-photochemical quenching. It was found that, whilst this capacity was species dependent, it did not depend upon the conditions under which the plant was grown. The possible role of zeaxanthin as a determinant of quenching capacity was examined by measuring the contents of xanthophyll cycle carotenoids for each species. Comparing species, no correlation was seen between the saturated level of non-photochemical quenching and zeaxanthin content expressed relative to either total carotenoid or to chlorophyll. When zeaxanthin was expressed relative to the amount of xanthophyll cycle intermediates (zeaxanthin, antheraxanthin and violaxanthin), a weak correlation was seen.     

Short-term down-regulation of zeaxanthin epoxidation in Arabidopsis thaliana in response to photo-oxidative stress conditions

The epoxidation of zeaxanthin (Zx) to violaxanthin after exposure to different light stress conditions has been studied in Arabidopsis (Arabidopsis thaliana). Formation of Zx was induced by illumination of intact leaves for up to 8 h at different light intensities and temperatures. The kinetics of epoxidation was found to be gradually retarded with increasing light stress during pre-illumination, indicating a gradual down-regulation of the Zx epoxidase activity. Retardation of the epoxidation rates by a factor of up to 10 was inducible either by increasing the light intensity or by extending the illumination time or by decreasing the temperature during pre-illumination. The retardation of the epoxidation kinetics was correlated with a decrease of the PSII quantum efficiency after the pre-illumination treatment. Experiments with the stn7/stn8 mutant of Arabidopsis indicated that the thylakoid protein kinases STN7 and STN8, which are required for the phosphorylation of PSII proteins, are not involved in the short-term down-regulation of Zx epoxidation. However, the retardation of Zx epoxidation was maintained in thylakoids isolated from pre-illuminated leaves, indicating that a direct modification of the Zx epoxidase is most likely involved in the light-induced down-regulation.     

Ferredoxin-quinone reductase benefits cyclic electron flow around photosystem 1 in tobacco leaves upon exposure to chilling stress under low irradiance

The function of chloroplast ferredoxin quinone reductase (FQR)-dependent flow was examined by comparing a wild type tobacco and a tobacco transformant (ΔndhB) in which the ndhB gene had been disrupted with their antimycin A (AA)-fed leaves upon exposure to chilling temperature (4 °C) under low irradiance (100 μmol m⁻² s⁻¹ photon flux density). During the chilling stress, the maximum photochemical efficiency of photosystem (PS) 2 (F_v/F_m) decreased markedly in both the controls and AA-fed leaves, and P700⁺ was also lower in AA-fed leaves than in the controls, implying that FQR-dependent cyclic electron flow around PS1 functioned to protect the photosynthetic apparatus from chilling stress under low irradiance. Under such stress, non-photochemical quenching (NPQ), particularly the fast relaxing NPQ component (q_f) and the de-epoxidized ratio of the xanthophyll cycle pigments, (A+Z)/(V+A+Z), formed the difference between AA-fed leaves and controls. The lower NPQ in AA-fed leaves might be related to an inefficient proton gradient across thylakoid membranes (ΔpH) because of inhibiting an FQR-dependent cyclic electron flow around PS1 at chilling temperature under low irradiance.     

Characteristics of Photosynthetic Apparatus in Mn-Starved Maize Leaves

The effects of Mn-deficiency on CO₂ assimilation and excitation energy distribution were studied using Mn-starved maize leaves. Mn-deficiency caused about 70 % loss in the photon-saturated net photosynthetic rate (P _N) compared to control leaves. The loss of P _N was associated with a strong decrease in the activity of oxygen evolution complex (OEC) and the linear electron transport driven by photosystem 2 (PS2) in Mn-deficienct leaves. The photochemical quenching of PS2 (q_P) and the maximum efficiency of PS2 photochemistry (F_v/F_m) decreased significantly in Mn-starved leaves under high irradiance, implicating that serious photoinhibition took place. However, the high-energy fluorescence quenching (q_E) decreased, which was associated with xanthophyll cycle. The results showed that the pool of de-epoxidation components of the xanthophyll cycle was lowered markedly owing to Mn deficiency. Linear electron transport driven by PS2 de-creased significantly and was approximately 70 % lower in Mn-deficient leaves than that in control, indicating less trans-thylakoid pH gradient was built in Mn deficient leaves. We suggest that the decrease of non-radiative dissipation depending on xanthophyll cycle in Mn-starved leaves is a result of the deficiency of trans-thylakoid pH gradient.     

Pigment apparatus in <Emphasis Type="Italic">Ajuga reptans</Emphasis> plants as affected by adaptation to light growth conditions

Mechanisms of adaptation of the photosynthetic apparatus at the level of pigment complex in a shade-tolerant bugle plant (Ajuga reptans L.) grown at full solar irradiation in an open plot were studied. In “sun” plants, the content of photosynthetic pigments decreased markedly as compared to “shade” plants grown under a forest canopy at 5–10% of the full solar irradiation. In leaves of sun plants, the portion of β-carotene and lutein in the carotenoid spectrum was higher than in shade plant leaves, antheraxanthin and zeaxanthin were present, and de-epoxidation of violaxanthin was by an order of magnitude higher in sun plant leaves reaching 40%. The data obtained indicate the role of the violaxanthin cycle in the protection of photosynthetic apparatus in a shade-tolerant plant against destruction under excessive irradiation.     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using ³¹P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L_β) to liquid-crystalline (L_α) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L_α to the inverted hexagonal (H_II) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L_α phase compared to those in the presence of H_II phase forming lipids. Our data furthermore imply that the H_II phase is better suited to maintain high VDE activities at lower temperatures.     

Strategies for the suppression of peroxidase gene expression in tobacco. II.In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs

Several strategies involving the use of antisense and ribozyme constructs in different expression vectors were investigated as methods of suppressing gene expressionin planta. We had previously identified an efficiently cleaving ribozyme (Rz), with two catalytic units and 60 nucleotide (nt) of complementary sequence, to the ligninforming peroxidase of tobacco (TPX). This Rz was cloned behind the 35S CaMV (35S) and nopaline synthase (NOS) promoters, and into a vector utilising the tobacco tyrosine tRNA for expression. For comparison with more traditional antisense strategies, full-length TPX antisense (AS) constructs were also constructed behind the NOS and 35S promoters. Populations of transgenic tobacco containing these constructs were produced and compared to control plants transformed with the vector only. Significant suppression of peroxidase expression in the range of 40–80% was seen in the T₀ and T₁ populations carrying 35S-AS, 35S-Rz and tRNA-Rz constructs. Co-segregation of the suppressed peroxidase phenotype and the tRNA-Rz transgenes was demonstrated. Northern blot analysis indicated that levels of TPX mRNA were lower in the Rz plants. No evidence of mRNA cleavage was observed and thus it was unclear if the Rz constructs were acting as Rzsin vivo. Transgenic plants containing the tRNA-Rz construct had significantly lower levels of peroxidase than the other transgenic plants. There was no significant difference in levels of suppression of TPX between the short Rz in the 35S vector and the full-length AS constructs. Although peroxidase levels were significantly reduced in transgenic plants carrying 35S-AS, 35S-Rz and tRNA-Rz constructs, no significant difference in lignin levels was observed.     

Reduced levels of cytochrome b 6/f in transgenic tobacco increases the excitation pressure on Photosystem II without increasing sensitivity to photoinhibition in vivo

We have examined tobacco transformed with an antisense construct against the Rieske-FeS subunit of the cytochromeb ₆ f complex, containing only 15 to 20% of the wild-type level of cytochrome f. The anti-Rieske-FeS leaves had a comparable chlorophyll and Photosystem II reaction center stoichiometry and a comparable carotenoid profile to the wild-type, with differences of less than 10% on a leaf area basis. When exposed to high irradiance, the anti-Rieske-FeS leaves showed a greatly increased closure of Photosystem II and a much reduced capacity to develop non-photochemical quenching compared with wild-type. However, contrary to our expectations, the anti-Rieske-FeS leaves were not more susceptible to photoinhibition than were wild-type leaves. Further, when we regulated the irradiance so that the excitation pressure on photosystem II was equivalent in both the anti-Rieske-FeS and wild-type leaves, the anti-Rieske-FeS leaves experienced much less photoinhibition than wild-type. The evidence from the anti-Rieske-FeS tobacco suggests that rapid photoinactivation of Photosystem II in vivo only occurs when closure of Photosystem II coincides with lumen acidification. These results suggest that the model of photoinhibition in vivo occurring principally because of limitations to electron withdrawal from photosystem II does not explain photoinhibition in these transgenic tobacco leaves, and we need to re-evaluate the twinned concepts of photoinhibition and photoprotection.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlophenyl)-1,-dimethylurea - Fo and Fo minimal fluorescence when all PS II reaction centers are open in dark- and light-acclimated leaves, respectively - Fm and Fm maximal fluorescence when all PS II reaction centers are closed in dark- and light-acclimated leaves, respectively - Fv variable fluorescence (Fm-Fo) in dark acclimated leaves - Fv variable fluorescence (Fm-Fo) in lightacclimated leaves - NPQ non-photochemical quenching of fluorescence - PS I and PS II Photosystem I and II - P680 primary electron donor of the reaction center of PS II - PFD photosynthetic flux density - Q_A primary acceptor quinone of PS II - q_p photochemical quenching of fluorescence - V+A+Z violaxanthin+antheraxanthin+zeaxanthin