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Role of histidines in the binding of violaxanthin de-epoxidase to the thylakoid membrane as studied by site-directed mutagenesis
Abstract
Regulation of violaxanthin de-epoxidase (VDE) involves a conformational change at low lumenal pH, followed by binding of the enzyme to the thylakoid membrane. The role of histidine residues in this process was studied by release of unbound enzyme from thylakoids upon sonication, on a pH scale from 4.7 to 7.1. The co-operativity for binding of spinach VDE (four histidines) to the membrane was found to be 3.8, with respect to protons, and had an inflexion point at pH 6.6, whereas VDE from wheat (three histidines) showed a co-operativity of 2.9 and had an inflexion point at pH 6.2. Mutant forms of VDE were constructed and probed for their binding to the outside of thylakoid membranes. With one or two histidines substituted for alanine or arginine, a lower co-operativity (1.6–2.3) was found, compared with the wild type. Based on these findings, and that the pKa value for histidine is within the range where the VDE binding takes place, we propose that protonation of the histidine residues at low pH induces the conformational change of VDE, and hence indirectly regulates binding of the enzyme to the thylakoid membrane.
... The optimal pH for spinach VDE activity is about 5 (Havir et al., 1997). Protonation of some amino acids residues of VDE in this species takes place cooperatively at 6.6 (Bratt et al., 1995; Gisselsson et al., 2004) and at 6.0 for wheat VDE (Gisselsson et al., 2004). This corresponds to different numbers of histidine residues in the VDE molecule (4 in spinach, 3 in wheat enzyme). ...
... The optimal pH for spinach VDE activity is about 5 (Havir et al., 1997). Protonation of some amino acids residues of VDE in this species takes place cooperatively at 6.6 (Bratt et al., 1995; Gisselsson et al., 2004) and at 6.0 for wheat VDE (Gisselsson et al., 2004). This corresponds to different numbers of histidine residues in the VDE molecule (4 in spinach, 3 in wheat enzyme). ...
... This corresponds to different numbers of histidine residues in the VDE molecule (4 in spinach, 3 in wheat enzyme). Protonation of histidine is suggested to be engaged in VDE binding to the membrane, not necessarily directly, but possibly by inducing conformational changes in the enzyme molecule (Gisselsson et al., 2004 ). Dicyclohexylcarbodiimide (DCCD) changes such pH dependence by shifting the structure alteration point of spinach VDE by 0.3 pH unit to more alkaline conditions, and it reduces the rate of the deepoxidation reaction, predominantly the second step (antheraxanthin – zeaxanthin transformation). ...
Lipocalins are a widely distributed group of proteins whose common feature is the presence of six-or eight-stranded beta-barrel in their tertiary structure and highly conservative motifs short conserved region, (SCR) in their amino acid sequences. The presence of three SCRs is typical for kernel lipocalins, while outlier lipocalins have only one or two such regions. Owing to their ability to bind and transport small, hydrophobic molecules, lipocalins participate in the distribution of such substances. However, the physiological significance of lipocalins is not limited to transfer processes. They play an important role in the regulation of immunological and developmental processes, and are also involved in the reactions of organisms to various stress factors and in the pathways of signal transduction. Of special interest is the enzymatic activity found in a few members of the lipocalin family, as well as the interaction with natural membranes, both directly with lipids and through membrane-localized protein receptors.
... VDE is localized in thylakoid lumen and its activity is highly dependent on lumen pH [50]. The apparent n H and pK a values for VDE protonation have been estimated in vitro to be 4.5 ± 0.5 and 6.25 ± 0.25 respectively [31,[51][52][53][54]. ...
... Z + A accumulation and PsbS protonation predicted very similar effective pK a values (∼ 6.8), but very different n H values (∼ 4.3 vs. ∼ 1.0 respectively). These fits conform to the expected relative Hill coefficients for the two processes [31,[51][52][53], partially validating our estimates. ...
... The pK a of protonation of VDE determined in vitro is between 6.0 and 6.5 [31,[51][52][53]. The apparent pK a for Z + A accumulation from our fit to Fig. 8A is shifted upwards from in vitro estimates by 0.3 to 0.8 units. ...
Endogenous probes of light-induced transthylakoid proton motive force (pmf), membrane potential (Deltapsi) and DeltapH were used in vivo to assess in Arabidopsis the lumen pH responses of regulatory components of photosynthesis. The accumulation of zeaxanthin and protonation of PsbS were found to have similar pK(a) values, but quite distinct Hill coefficients, a feature allowing high antenna efficiency at low pmf and fine adjustment at higher pmf. The onset of "energy-dependent' exciton quenching (q(E)) occurred at higher lumen pH than slowing of plastoquinol oxidation at the cytochrome b(6)f complex, presumably to prevent buildup of reduced electron carriers that can lead to photodamage. Quantitative comparison of intrinsic probes with the electrochromic shift signal in situ allowed quantitative estimates of pmf and lumen pH. Within a degree of uncertainly of approximately 0.5 pH units, the lumen pH was estimated to range from approximately 7.5 (under weak light at ambient CO(2)) to approximately 5.7 (under 50 ppm CO(2) and saturating light), consistent with a 'moderate pH' model, allowing antenna regulation but preventing acid-induced photodamage. The apparent pK(a) values for accumulation of zeaxanthin and PsbS protonation were found to be approximately 6.8, with Hill coefficients of about 4 and 1 respectively. The apparent shift between in vitro violaxanthin deepoxidase protonation and zeaxanthin accumulation in vivo is explained by steady-state competition between zeaxanthin formation and its subsequent epoxidation by zeaxanthin epoxidase. In contrast to tobacco, Arabidopsis showed substantial variations in the fraction of pmf (0.1-0.7) stored as Deltapsi, allowing a more sensitive qE response, possible as an adaptation to life at lower light levels.
... The light-dependent build-up of the transthylakoidal proton gradient (DpH) and the subsequent acidification of the lumen is necessary for the binding of the de-epoxidase to the thylakoid membrane in order to get access to its xanthophyll substrate [8,9]. This process is regulated by the protonation of a glutamic acid-rich domain located in the highly charged C-terminal part of the enzyme and by the protonation of histidine residues located in the lipocalin region [11][12][13]. In diatoms, there are two XCs [9,10,14], one of them is identical to the XC found in higher plants, performing the de-epoxidation of violaxanthin (Vx) to zeaxanthin (Zx) via the intermediate antheraxanthin by the violaxanthin de-epoxidase (VDE). ...
... VDL and VDR enzymes might have a different localization and functional role than DDE as indicated by the differential lightinduced expression of the Vdl genes compared to the Dde (or Vde) gene [15] and by their putative lower ability to be regulated by protonation as suggested by their differential amino acid content [15,16]. Therefore, it must be assumed that while the activity of VDL and VDR enzymes might still be controlled by protonation as DDE [8,[11][12][13], they most probably have a different/lower reactivity to pH changes. As a consequence, the VDLs and VDRs probably lack an essential feature for their involvement in the XC, at least the way DDE is involved in the XC, which is the fine regulation of their activity as a function of the lumen acidification. ...
Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms.
... The VDE has been characterized as a water soluble protein localized in the thylakoid lumen [6] and the pH-dependent binding to (at low pH) and release from (at high pH) the thylakoid membrane is thought to be central to the regulation of VDE activity [9]. Binding of the VDE to the thylakoid membrane occurs most likely through the charged C-terminal domain of the enzyme and an important role of histidine residues in the binding process has been proposed [123]. Since the co-operativity for binding of VDE, with respect to protons, and the pH value of the respective inflexion point was found to be strongly dependent on the numbers of histidine residues, it was proposed that protonation of the histidine residues at low pH induces a conformational change of the VDE, and by that contributes to the regulation of binding of VDE to the thylakoid membrane [123]. ...
... Binding of the VDE to the thylakoid membrane occurs most likely through the charged C-terminal domain of the enzyme and an important role of histidine residues in the binding process has been proposed [123]. Since the co-operativity for binding of VDE, with respect to protons, and the pH value of the respective inflexion point was found to be strongly dependent on the numbers of histidine residues, it was proposed that protonation of the histidine residues at low pH induces a conformational change of the VDE, and by that contributes to the regulation of binding of VDE to the thylakoid membrane [123]. A similar role has earlier been postulated for glutamic acid residues, which are located in the C-terminal domain of the VDE [36,39]. ...
The violaxanthin cycle describes the reversible conversion of violaxanthin to zeaxanthin via the intermediate antheraxanthin. This light-dependent xanthophyll conversion is essential for the adaptation of plants and algae to different light conditions and allows a reversible switch of photosynthetic light-harvesting complexes between a light-harvesting state under low light and a dissipative state under high light. The photoprotective functions of zeaxanthin have been intensively studied during the last decade, but much less attention has been directed to the mechanism and regulation of xanthophyll conversion. In this review, an overview is given on recent progress in the understanding of the role of (i) xanthophyll binding by antenna proteins and of (ii) the lipid properties of the thylakoid membrane in the regulation of xanthophyll conversion. The consequences of these findings for the mechanism and regulation of xanthophyll conversion in the thylakoid membrane will be discussed.
... After truncation of the C-terminal domain the membrane binding ability was lost, indicating that this domain is involved in membrane binding (Hieber et al. 2002). The ability of VDE to bind to the thylakoid membrane has been shown to be closely regulated by pH and occurs at a pH similar to the pH of where VDE shows the highest activity (Gisselsson et al. 2004). The pH-dependent regulation of VDE is of great importance to the plants and algae since it will partly set the level of light intensity that will be tolerated before zeaxanthin-dependent light quenching begins. ...
Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.
... The structural analysis of the pH-dependent conformational transition described above suggests that His-121 should play a key role in initiating the opening of the lipocalin barrel prior to protein dimerization. Since His pK a values are ideal for pH sensor activity in VDE, such a role for His residues has previously been suggested and studied using site-directed mutagenesis and chemical modification (Emanuelsson et al., 2003;Gisselsson et al., 2004). Several pairwise substitutions and only one single mutation (His-124) were performed, revealing a complete loss of VDE activity when all four His residues were substituted with Ala or Arg. ...
Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent K m that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDE cd) at acidic and neutral pH. At neutral pH, VDE cd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin b-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.
... Zeaxanthin accumulation, thus, must be finely regulated in photosynthetic organisms to reach the optimal balance between the contrasting necessities of protection from high irradiation damage and efficient light harvesting under limiting illumination. It is well known that VDE is regulated by lumenal pH and when its value decreases below 6 the protein is activated thanks to a conformational change [12,34,35]. Additional possible mechanisms for VDE activity regulation were not identified. ...
When exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme Violaxanthin De-Epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation.
Copyright © 2015. Published by Elsevier B.V.
... Site directed mutagenesis and chemical modifications [11,19] did suggest that a His residue plays a key role in pH-dependent conformation changes, since it ensures that the pKa values are ideal for pH sensor activity [11,20,21]. Some conserved residues in the active site have also been studied, such as a hydrogen bondstabilized dimer in VDE, salt bridges, and several charged or polar residues ( Fig. 1 black squares) [11]. ...
Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE) from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2(nd) hour and the 9(th) day, respectively. The ratio of (A+Z)/(V+A+Z) and non-photochemical quenching (NPQ) is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII) in transgenic Arabidopsis under high light stress.
... In H + domains sequestered within the proteo-lipid core of the thylakoid membrane (Ewy and Dilley 2000) protonation of E122 and E226 of PsbS supports zeaxanthin binding (Haripal et al. 2006) and facilitates energy transfer between pigment protein complexes (Niyogi et al. 2005). Lumen acidification also regulates protonation of histidine residues in violaxanthin de-epoxidase (VDE) (Emanuelsson et al. 2003) and regulates binding of VDE to the thylakoids (Gisselsson et al. 2004), where it reduces epoxidized end-group moieties in xanthophylls (Eskling et al. 1997). In addition, below pH 5.3, reduced plastocyanin performs structural changes at the metal binding site, leading to reversible inhibitory aggregation of the electron transmitter protein (Sas et al. 2006). ...
Plants sense environmental signals by various sensory mechanisms distributed over all subcellular compartments. Plasma membrane
integrated receptors sense pathogens, excess salinity, hormonal signals and light. Through membrane transporters, soluble
environmental signals are imported into the cytosol, where the ion, redox and pH homeostasis is controlled. Endomembranes
and microbodies form small cellular sub-compartments involved in environmental sensing and signal transduction. Light sensing,
which strongly controls plant metabolism, is separated into qualitative light sensing by photoreceptor proteins at the plasma
membrane and in the cytosol and quantitative light sensing through photosynthesis. Embedded into the cytosol, besides light
sensing, the organelles mainly integrate environmental signals, respond to cytosolic changes and trigger metabolite fluxes,
the energy supply and developmental programmes such as cell death. Covering a selection of environmental sensing mechanisms,
recent insights into the diversity of subcellular sites and mechanisms of environmental sensing will be reviewed.
... Xanthophyll cycle-dependent nonradiative energy dissipation may be involved in the improvement of GB: The photosynthetic apparatus could protect itself under stress by increasing the xanthophyll cycle around PSI and PSII to dissipate excessive energy (Havaux et al. 2000). The xanthophyll cycle plays a major role in photo-protection and the resulting zeaxanthin plays a key role in NPQ (Gisselsson et al. 2004). Based on the results above (Figs. ...
To investigate the role of glycine betaine in photosynthesis under stress, a transgenic wheat (Triticum aestivum L.) line T6 overaccumulating glycine betaine and its wild type Shi4185 were used. Seedlings were exposed to conditions of
drought (30%, PEG-6000), heat (40°C) and their combination. The results revealed ultrastructural damage to the chloroplast
and thylakoid lamellae with the withered phenotype by both drought and heat stress, and the damage was exacerbated by the
combination of drought and heat. The appearance of a K step in the typical O-J-I-P curve and the decrease of Hill activity
indicated a reduction of oxygen evolving complex function caused by stress. The greater damage was found in wild type than
T6. Overaccumulation of glycine betaine in T6 could protect lipids in the thylakoid membrane from damage and stabilize the
index of unsaturated fatty acids under stress. A lower ratio of monogalactosyl diacylglycerol/digalactosyl diacylglycerol
and higher phosphatidylglycerol content in the thylakoid membrane of T6 were also observed under stress. These effects can
promote stability of the thylakoid membrane. Otherwise, glycine betaine overaccumulation decreased photoinhibition of PSII
under stress. The results also suggest that xanthophyll cycle-dependent non-radiative energy dissipation may be involved in
the GB-mediated effects on PSII function under stress conditions.
Additional key wordschloroplast ultrastructure-fatty acids-Hill activity-lipids-nonradiative energy dissipation-oxygen-evolving complex-thylakoid membrane
... Produced NADPH (p) was estimated through laboratory measures, while pH value was deduced by combining data from literature [7,14,29] and applying the rate of change to the estimated pH values during the NPQ measure. VDE state (x, y) was set in relation to various pH values [9]. The change over time of violaxanthin (v) and zeaxanthin (z) was obtained with lab measurements during VDE activity. ...
A workflow for data analysis is introduced to synthesize flux regulation maps of a Metabolic P system from time series of data observed in laboratory. The procedure is successfully tested on a significant case study, the photosynthetic phenomenon called NPQ, which determines plant accommodation to environmental light. A previously introduced MP model of such a photosynthetic process has been improved, by providing an MP system with a simpler regulative network that reproduces the observed behaviors of the natural system. Two regression techniques were employed to find out the regulation maps, and interesting experimental results came out in the context of their residual analysis for model validation.
... VDE activity in lumenal chloroplast fractions [2] was assayed in citrate-phosphate buffer, pH 5.2, following published protocols [42,43] in the presence and absence of reduced E. coli Trx [26]. Proteolytic activities in the lumenal fraction and the influence of the thiol redox state on these activities were studied using the E. coli NTR/Trx system as a reductant [26]. ...
The light-dependent regulation of stromal enzymes by thioredoxin (Trx)-catalysed disulphide/dithiol exchange is known as a classical mechanism for control of chloroplast metabolism. Recent proteome studies show that Trx targets are present not only in the stroma but in all chloroplast compartments, from the envelope to the thylakoid lumen. Trx-mediated redox control appears to be a common feature of important pathways, such as the Calvin cycle, starch synthesis and tetrapyrrole biosynthesis. However, the extent of thiol-dependent redox regulation in the thylakoid lumen has not been previously systematically explored. In this study, we addressed Trx-linked redox control in the chloroplast lumen of Arabidopsis thaliana. Using complementary proteomics approaches, we identified 19 Trx target proteins, thus covering more than 40% of the currently known lumenal chloroplast proteome. We show that the redox state of thiols is decisive for degradation of the extrinsic PsbO1 and PsbO2 subunits of photosystem II. Moreover, disulphide reduction inhibits activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase, which participates in thermal dissipation of excess absorbed light. Our results indicate that redox-controlled reactions in the chloroplast lumen play essential roles in the function of photosystem II and the regulation of adaptation to light intensity.
... The structural analysis of the pH-dependent conformational transition described above suggests that His-121 should play a key role in initiating the opening of the lipocalin barrel prior to protein dimerization. Since His pK a values are ideal for pH sensor activity in VDE, such a role for His residues has previously been suggested and studied using site-directed mutagenesis and chemical modification (Emanuelsson et al., 2003; Gisselsson et al., 2004). Several pairwise substitutions and only one single mutation (His-124) were performed, revealing a complete loss of VDE activity when all four His residues were substituted with Ala or Arg. ...
Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin beta-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.
... The conserved core domains (about 250 amino acids) of the VDE-related proteins display only 15–20% identity with VDE and therefore were not included in the phylogenetic analyses. In particular, a number of amino acid positions that have been identified to be important for catalytic function of VDE (Emanuelsson et al. 2003; Gisselsson et al. 2004) are not conserved in the VDE-like proteins (data not shown). Moreover, in preliminary analyses, we could prove the catalytic function of the recombinant VDE protein from P. tricornutum, and initial mutational studies of the enzyme suggest that the replacement of even short motifs in the lipocalin domain is likely to result in an inactive enzyme (Nowoisky J, Herwig S, Lohr M, unpublished data). ...
Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.
... When overexcited, the lumenal pH of thylakoids drops to values below 6. This causes a conformational change of the enzyme, violaxanthin deepoxidase (VDE) presumably through protonation of conserved histidines [1]. Subsequently, VDE docks to the lumenal side of the membrane and starts to convert violaxanthin to zeaxanthin through the intermediate antheraxanthin using ascorbic acid as a co-substrate. ...
Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 degrees C, 25 degrees C and 37 degrees C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H(II)), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L(alpha)), such as alpha-tocopherol and 8-cetylether (C(16)EO(8)), only 55% and 35% of the violaxanthin was converted at 25 degrees C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.
... This conversion is catalysed by the enzyme, violaxanthin de-epoxidase (VDE) which requires low pH, ascorbic acid as a co-substrate and lipids that can form inverted hexagonal phase [2] such as monogalactosyldiacylglycerol (MGDG). When the lumenal pH of thylakoids, upon overexcitation, drops to values below 6, a conformational change of VDE occurs presumably through protonation of conserved histidines [3]. VDE then docks to the membrane and starts converting violaxanthin to zeaxanthin via the intermediate antheraxanthin. ...
Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach (Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 degrees C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.
In recent years, with the global climate change, the intensity, frequency and duration of drought have increased significantly, which has become the main limiting factor for agricultural development in many areas. Glycine betaine (GB) is an effective stress-resistant substance. In this experiment, the effects of the genetic engineering of GB synthesis on photosynthetic apparatus of tobacco under drought stress were studied using transgenic tobacco (T) accumulating GB and wild-type tobacco (K326, WT). Potted tobaccos were subjected to drought stress (controlled irrigation, 25 °C ± 1 °C, a relative humidity: 75–80%) for 9 days, photosynthetic gas exchange parameters, chlorophyll a fluorescence, structure of chloroplast and thylakoid membrane, and protein function of thylakoid membrane were examined under different drought stress time (days). The results showed that T tobacco could accumulate GB and the accumulated GB improved the resistance of the photosynthetic apparatus to drought stress. Under drought stress, the damage of chloroplast and thylakoid lamellae in T tobacco was less than that in WT tobacco, the accumulation of GB in T tobacco could maintain the stability of thylakoid membrane, improved the unsaturated fatty acid index (IUFA) of thylakoid membrane lipid, increased the contents of digalactosyl diacylglycerol (DGDG) and phosphatidylglycerol (PG), and decreased the ratio of monogalactosyl diaylglycerol (MGDG) to DGDG. In addition, under drought stress, the accumulation of GB in T tobacco alleviated the photo-inhibition of PSII, and the increase of xanthophyll cycle de-epoxidation may be one of the reasons for the enhancement of PSII function.
The xanthophyll cycle is the metabolic process by which the carotenoid violaxanthin is de‐epoxidated to zeaxanthin, a xanthophyll with a crucial photoprotective role in higher plants and mosses. The role of zeaxanthin is still unclear in green algae, and a peculiar violaxanthin de‐epoxidating enzyme was found in the model organism Chlamydomonas reinhardtii. Here, we investigated the molecular details and functions of the xanthophyll cycle in the case of Chlorella vulgaris, one of the green algae most considered for industrial cultivation, where resistance to high light stress is a prerequisite for sustainable biomass production.
Identification of the violaxanthin de‐epoxidase enzyme in C. vulgaris was performed by genome mining and in vitro analysis of the catalytic activity of the gene product identified. The photoprotective role of zeaxanthin was then investigated in vivo and in isolated pigment‐binding complexes.
The results obtained demonstrate the functioning, even though with a different pH sensitivity, of a plant‐like violaxanthin de‐epoxidase enzyme in C. vulgaris. Differently from C. reinhardtii, zeaxanthin accumulation in C. vulgaris was found to be crucial for photoprotective quenching of excitation energy harvested by both photosystem I and II.
These findings demonstrate an evolutionary divergence of photoprotective mechanisms among Chlorophyta.
Glycine betaine (GB) is an effective compatible solute that improves the tolerance in plants to various stresses. We investigated
the effects of 2 mM GB applied to the roots of a tobacco (Nicotiana tabacum L.) cultivar on enhancing photosynthesis under low-temperature (LT) stress (5/5 °C, 12/12 h, 300 µmol m−2 s−1) and in the subsequent recovery (25/18 °C) from the stress. The net photosynthetic rate, intrinsic efficiency measured as
the ratio of variable to maximum fluorescence, and actual efficiency of the photochemistry of photosystem 2 as well as the
ATPase activity in the thylakoid membrane decreased, and a distinct K step in the fluorescence transient O-J-I-P appeared
under cold stress. Exogenous GB alleviated the decrease in all these parameters. The LT-stress induced the accumulation of
33–66 kDa polypeptides and decreased the proportion of unsaturated fatty acids in the thylakoid membrane. In plants subjected
to LT-stress, GB protected these polypeptides from damage and enhanced the proportion of unsaturated fatty acids. An increase
in non-radiative energy dissipation (NPQ) may be involved in the improvement of the function of the thylakoid membrane by
GB since exogenous GB protected violaxanthin de-epoxidase and enhanced NPQ.
In this paper we apply the formalism of metabolic P systems for modeling an important phenomenon of photochemical organisms,
which determines the plants accommodation to the environmental light. By using some experimental data of this phenomenon,
we determine an MP system which discovers, in a specific simplified case, the regulation mechanism underling the non photochemical
quenching phenomenon and reproduces, with a good approximation, the observed behavior of the natural system.
Photosynthesis is the process used by plants, algae and some bacteria to obtain biochemical energy from sunlight. It is the
most important process allowing life on earth. In this work, by applying the Log Gain theory of Metabolic P Systems, we define
a mathematical model of an important photosynthetic phenomenon, called Non Photochemical Quenching (shortly NPQ), that determines
the plant accommodation to the environmental light. Starting from experimental data of this phenomenon, we are able to deduce
a Metabolic P system which provides, in a specific simplified case, the regulation mechanism underling the NPQ process. The
dynamics of our model, generated by suitable computational tools, reproduce, with a very good approximation, the observed
behaviour of the natural system.
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.
Plants and algae often absorb too much light-more than they can actually use in photosynthesis. To prevent photo-oxidative damage and to acclimate to changes in their environment, photosynthetic organisms have evolved direct and indirect mechanisms for sensing and responding to excess light. Photoreceptors such as phototropin, neochrome, and cryptochrome can sense excess light directly and relay signals for chloroplast movement and gene expression responses. Indirect sensing of excess light through biochemical and metabolic signals can be transduced into local responses within chloroplasts, into changes in nuclear gene expression via retrograde signaling pathways, or even into systemic responses, all of which are associated with photoacclimation.
Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 mM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).
The harnessing of solar energy by photosynthesis depends on a safety valve that effectively eliminates hazardous excess energy and prevents oxidative damage to the plant cells. Many of the compounds that protect plant cells also protect human cells. Improving plant resistance to stress may thus have the beneficial side effect of also improving the nutritional quality of plants in the human diet. The pathways that synthesize these compounds are becoming amenable to genetic manipulation, which may yield benefits as widespread as improved plant stress tolerance and improved human physical and mental health.
Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on Km suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.
Zeaxanthin has been correlated with high-energy non-photochemical fluorescence quenching but whether antheraxanthin, the intermediate in the pathway from violaxanthin to zeaxanthin, also relates to quenching is unknown. The relationships of zeaxanthin, antheraxanthin and ΔpH to fluorescence quenching were examined in chloroplasts ofPisum sativum L. cv. Oregon andLactuca sativa L. cv. Romaine. Data matrices as five levels of violaxanthin de-epoxidation against five levels of light-induced lumen-proton concentrations were obtained for both species. The matrices included high levels of antheraxanthin as well as lumen-proton concentrations induced by subsaturating to saturation light levels. Analyses of the matrices by simple linear and multiple regression showed that quenching is predicted by models where the major independent variable is the product of lumen acidity and de-epoxidized xanthophylls, the latter as the sum of zeaxanthin and antheraxanthin. The interactions of lumen acidity and xanthophyll concentration are shown in three-dimensional plots of the best-fit multiple regression models. Antheraxanthin apparently contributes to quenching as effectively as zeaxanthin and explains quenching previously not accounted for by zeaxanthin. Hence, we propose that all high-energy dependent quenching is xanthophyll dependent. Quenching requires a threshold lumen pH that varies with xanthophyll composition. After the threshold, quenching is linear with lumen acidity or xanthophyll composition.
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 Km 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 Km±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.
Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH4)2SO4 fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 μmol g−1 s−1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with β-mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b
6 and an internal sequence of polyphenol oxidase.
The cleavage of disulfide bridges is of importance in both functional and structural studies of proteins. Of the three available methods for accomplishing this—namely, oxidation, sulfitolysis and reduction, the third procedure is often the method of choice since the first method suffers from lack of reversibility as well as undesirable side reactions, and the second from the difficulty in achieving complete reaction. While β-mercaptoethanol has been extensively used for both selective and complete reduction of disulfide bridges, it can be replaced by dithiothreitol (DTT). This reagent can be used at a much lower concentration than β-mercaptoethanol by virtue of its lower oxidation-reduction potential and its resistance to air oxidation compared to β-mercaptoethanol. This can be of particular importance when radioactive alkylating agents are to be employed, since only a very small molar excess of DTT is required for complete reduction and therefore only minimal quantities of radioactive material need be wasted in alkylating excess reducing agent.
The violaxanthin de-epoxidase (VDE) gene from spinach (Spinacia oleracea) was cloned, sequenced (GenBank AJ 250433), and expressed in Escherichia coli. The highest obtained conversion rate of violaxanthin was 86 nmol s−1 per litre of growth medium, corresponding to an amount of active enzyme of 0.4 mg l−1. Sequence comparison between VDE from different species were made and particular interest was focused on four highly conserved histidines (H121,124,167,173) and their possible involvement in enzymatic activity. Chemical modification of the histidines using DEPC or by site-directed mutations resulted in partial or total inactivation of the enzyme. The chemical modification could be reversed by hydroxylamine treatment, regenerating a large percentage of the original activity. The histidine residues, which are located in pairs close to each other, were pairwise substituted for either alanine or arginine. This resulted in one inactive mutant (H121,124R) and three mutants with very different activities and decreased binding of ascorbic acid, as reflected by an up to four-fold increase in Km. A substitution of all four histidines for either alanine or arginine resulted in inactive enzymes. Based on these results it is suggested that the histidine residues are important for the activity of VDE.
The xanthophyll cycle is of great importance in relation to light stress. Particularly, interest has been focused on the possible
photoprotective role of zeaxanthin. In higher plants under light stress, zeaxanthin is formed from violaxanthin in a reaction
catalyzed by violaxanthin de-epoxidase (VDE). The reverse reaction is catalyzed by zeaxanthin epoxidase (ZE) under low light
or in darkness. VDE has been purified from spinach and lettuce as a 43-kDa protein. The gene has been cloned and sequenced
from several species, and a few mutants have been isolated. The gene is nuclear encoded and the transit peptide is characteristic
for targeting to the thylakoid lumen. The activity of VDE is affected by factors such as a pH-dependent binding to the thylakoid
membrane, concentration of ascorbic acid, temperature and availability of violaxanthin in relation to amount, type and distribution
of pigment-protein complexes in the membrane. The information about ZE is more limited. The enzyme has not yet been isolated
but its gene has been cloned and sequenced and a number of mutants have been isolated. The role of the xanthophyll cycle in
the dissipation of excess light energy will be discussed particularly in relation to the recent progress in studies on various
mutants. The possible role of the xanthophyll cycle in other processes, such as protection against oxidative stress of lipids,
regulation of membrane fluidity, participation in blue light responses, and regulation of abscisic acid synthesis will also
be presented.
Zeaxanthin, a carotenoid in the xanthophyll cycle, has been suggested to play a role in the protection against photodestruction. We have studied the importance of the parameters involved in zeaxanthin formation by comparing spinach plants grown in low light (100 to 250 mol m-2 s-1) to plants transferred to high light (950 mol m-2 s-1). Different parameters were followed for a total of 11 days. Our experiments show that violaxanthin de-epoxidase decreased between 15 and 30%, the quantity of xanthophyll cycle pigments doubled to 100 mmol (mol Chl)-1, corresponding to 27 mol m-2, and the rate of violaxanthin to zeaxanthin conversion was doubled. Lutein and neoxanthin increased from 50 to 71 mol m-2 and from 16 to 23 mol m-2, respectively. On a leaf area basis, chlorophyll and -carotene levels first decreased and then after 4 days increased. The chlorophyll a/b ratio was unchanged. The quantity of ascorbate was doubled to 2 mmol m-2, corresponding to an estimated increase in the chloroplasts from 25 to 50 mM. In view of our data, we propose that the increase in xanthophyll cycle pigments and ascorbate only partly explain the increased rate of conversion of violaxanthin to zeaxanthin, but the most probable explanation of the faster conversion is an increased accessibility of violaxanthin in the membrane.
Using DTT and iodoacetamide as a novel irreversible method to inhibit endogenous violaxanthin de-epoxidase, we found that violaxanthin could be converted into zeaxanthin from both sides of the thylakoid membrane provided that purified violaxanthin de-epoxidase was added. The maximum conversion was the same from both sides of the membrane. Temperature was found to have a strong influence both on the rate and degree of maximal violaxanthin to zeaxanthin conversion. Thus only 50% conversion of violaxanthin was detected at 4 C, whereas at 25 C and 37 C the degree of conversion was 70% and 80%, respectively. These results were obtained with isolated thylakoids from non-cold acclimated leafs. Pigment analysis of sub-thylakoid membrane domains showed that violaxanthin was evenly distributed between stroma lamellae and grana partitions. This was in contrast to chlorophyll a and -carotene which were enriched in stroma lamellae fractions while chlorophyll b, lutein and neoxanthin were enriched in the grana membranes. In combination with added violaxanthin de-epoxidase we found almost the same degree of conversion of violaxanthin to zeaxanthin (73–78%) for different domains of the thylakoid membrane. We conclude that violaxanthin de-epoxidase converts violaxanthin in the lipid matrix and not at the proteins, that violaxanthin does not prefer one particular membrane region or one particular chlorophyll protein complex, and that the xanthophyll cycle pigments are oriented in a vertical manner in order to be accessible from both sides of the membrane when located in the lipid matrix.
Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on Km suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.
Recent studies have provided new insights into the ways that plants may dissipate excess photons and electrons, thereby protecting the photosynthetic apparatus against light-induced damage. These 'safety valves' include nonphotochemical mechanisms for quenching excited chlorophylls, as well as alternative electron acceptors such as oxygen.
Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure flanked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A full-length Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N- and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the specific activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without affecting activity. The difficulties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13- to 19-fold increase in VDE specific activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identified a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the first reported example of plants with an increased level of VDE activity.
The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.
Bilayer-forming lipids were shown to be ineffective in sustaining the enzymatic activity of violaxanthin de-epoxidase. On the other hand, non-bilayer-forming lipids, regardless of their different chemical character, ensured high activity of violaxanthin de-epoxidase, resulting in conversion of violaxanthin to zeaxanthin. Our data indicates that the presence of lipids forming reversed hexagonal structures is necessary for violaxanthin de-epoxidase activity and this activity is dependent on the degree of unsaturation of the fatty acids. The significance of the reversed hexagonal phase domains in the conversion of violaxanthin into zeaxanthin in model systems and in the native thylakoid membranes is discussed.
Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O(2), 0% CO(2)) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F(v), or maximum fluorescence, F(m), and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of beta-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O(2) and 0% CO(2) resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F(v)/F(m), determined with the modulated fluorescence technique at room temperature, and the photon yield of O(2) evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.
Violaxanthin accessibility and temperature dependency for de-epoxidation in spinach thylakoid mem-branes Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentrations Molecular cloning of violax-anthin de-epoxidase from romaine lettuce and expression in Escherichia coli
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- H Stefa´
- P-A ˚ Albertsson
- H-E Bratt Ce
- P-O Arvidsson
- A M Carlsson
- H-E Rc Yamamoto
- Hy
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Photoinhibi-tion and zeaxanthin formation in intact leaves Antioxidants in photo-synthesis and human nutrition
- B K Winter
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- F-C Czygan
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Non-photochemical quenching. A response to excess light energy Safety valves for photosynthesis The pH dependence of violaxanthin deepoxidation in isolated pea chloroplasts
- P Li
- X-P Niyogi
- Ee Pfu¨
- Dilley
P, Li X-P, Niyogi KK (2001) Non-photochemical quenching. A response to excess light energy. Plant Physiol 125: 1558–1566 Niyogi KK (2000) Safety valves for photosynthesis. Curr Opin Plant Biol 3: 455–460 Pfu¨ EE, Dilley RA (1993) The pH dependence of violaxanthin deepoxidation in isolated pea chloroplasts. Plant Physiol 101: 65–71
Regulation of Photosynthesis
- Eskling M
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