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Hans-erik Åkerlund - One of the best experts on this subject based on the ideXlab platform.

  • Molecular studies on structural changes and oligomerisation of Violaxanthin De-Epoxidase associated with the pH-dependent activation
    Photosynthesis Research, 2016
    Co-Authors: Erik Ingmar Hallin, Kuo Guo, Mahmudul Hasan, Hans-erik Åkerlund
    Abstract:

    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.

  • Membrane curvature stress controls the maximal conversion of Violaxanthin to zeaxanthin in the Violaxanthin cycle--influence of alpha-tocopherol, cetylethers, linolenic acid, and temperature.
    Biochimica et biophysica acta, 2007
    Co-Authors: Anna Szilágyi, Marianne Sommarin, Hans-erik Åkerlund
    Abstract:

    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.

  • role of histidines in the binding of Violaxanthin de epoxidase to the thylakoid membrane as studied by site directed mutagenesis
    Physiologia Plantarum, 2004
    Co-Authors: Anna Gisselsson, Anna Szilágyi, Hans-erik Åkerlund
    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.

  • Violaxanthin de epoxidase the xanthophyll cycle enzyme requires lipid inverted hexagonal structures for its activity
    Biochemistry, 2004
    Co-Authors: Dariusz Latowski, Hans-erik Åkerlund, Kazimierz Strzalka
    Abstract:

    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.

  • Chemical and mutational modification of histidines in Violaxanthin De-Epoxidase from Spinacia oleracea
    Physiologia Plantarum, 2003
    Co-Authors: Anna Emanuelsson, Marie Eskling, Hans-erik Åkerlund
    Abstract:

    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. (Less)

Harry Y. Yamamoto - One of the best experts on this subject based on the ideXlab platform.

  • overexpression of Violaxanthin de epoxidase properties of c terminal deletions on activity and ph dependent lipid binding
    Planta, 2002
    Co-Authors: David A Hieber, Robert C. Bugos, Amy S. Verhoeven, Harry Y. Yamamoto
    Abstract:

    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 non-photochemical 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.

  • Plant lipocalins: Violaxanthin De-Epoxidase and zeaxanthin epoxidase.
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Arleen D. Hieber, Robert C. Bugos, Harry Y. Yamamoto
    Abstract:

    Abstract Violaxanthin De-Epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids Violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the Violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.

  • Antisense suppression of Violaxanthin De-Epoxidase in tobacco does not affect plant performance in controlled growth conditions.
    Photosynthesis research, 2000
    Co-Authors: Sue-hwei Chang, Robert C. Bugos, Wen-hao Sun, Harry Y. Yamamoto
    Abstract:

    Violaxanthin De-Epoxidase (VDE) catalyzes the de-epoxidation of Violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of Violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m−2 s−1 for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of Violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of Violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m−2 s−1 under controlled growth conditions as compared to wild-type tobacco.

  • Developmental Expression of Violaxanthin De-Epoxidase in Leaves of Tobacco Growing under High and Low Light
    Plant physiology, 1999
    Co-Authors: Robert C. Bugos, Sue-hwei Chang, Harry Y. Yamamoto
    Abstract:

    Violaxanthin De-Epoxidase (VDE) is a lumen-localized enzyme that catalyzes the de-epoxidation of Violaxanthin in the thylakoid membrane upon formation of a transthylakoid pH gradient. We investigated the developmental expression of VDE in leaves of mature tobacco (Nicotiana tabacum) plants grown under high-light conditions (in the field) and low-light conditions (in a growth chamber). The difference in light conditions was evident by the increased pool size (Violaxanthin + antheraxanthin + zeaxanthin, VAZ) throughout leaf development in field-grown plants. VDE activity based on chlorophyll or leaf area was low in the youngest leaves, with the levels increasing with increasing leaf age in both high- and low-light-grown plants. However, in high-light-grown plants, the younger leaves in early leaf expansion showed a more rapid increase in VDE activity and maintained higher levels of VDE transcript in more leaves, indicating that high light may induce greater levels of VDE. VDE transcript levels decreased substantially in leaves of mid-leaf expansion, while the levels of enzyme continued to increase, suggesting that the VDE enzyme does not turn over rapidly. The level of VDE changed in an inverse, nonlinear relationship with respect to the VAZ pool, suggesting that enzyme levels could be indirectly regulated by the VAZ pool.

  • xanthophyll cycle enzymes are members of the lipocalin family the first identified from plants
    Journal of Biological Chemistry, 1998
    Co-Authors: Robert C. Bugos, David A Hieber, Harry Y. Yamamoto
    Abstract:

    Abstract Violaxanthin De-Epoxidase and zeaxanthin epoxidase catalyze the addition and removal of epoxide groups in carotenoids of the xanthophyll cycle in plants. The xanthophyll cycle is implicated in protecting the photosynthetic apparatus from excessive light. Two new sequences for Violaxanthin De-Epoxidase from tobacco andArabidopsis are described. Although the mature proteins are well conserved, the transit peptides of these proteins are divergent, in contrast to transit peptides from other proteins targeted to the thylakoid lumen. Sequence analyses of both Violaxanthin De-Epoxidase and zeaxanthin epoxidase establish the xanthophyll cycle enzymes as members of the lipocalin family of proteins. The lipocalin family is a diverse group of proteins that bind small hydrophobic (lipophilic) molecules and share a conserved tertiary structure of eight β-strands forming a barrel configuration. This is the first reported identification of lipocalin proteins in plants.

Krishna K. Niyogi - One of the best experts on this subject based on the ideXlab platform.

  • Evolution of an atypical De-Epoxidase for photoprotection in the green lineage
    Nature plants, 2016
    Co-Authors: Zhirong Li, Graham Peers, Rachel M. Dent, Scarlett Y. Yang, Wiebke Apel, Lauriebeth Leonelli, Krishna K. Niyogi
    Abstract:

    Plants, algae and cyanobacteria need to regulate photosynthetic light harvesting in response to the constantly changing light environment. Rapid adjustments are required to maintain fitness because of a trade-off between efficient solar energy conversion and photoprotection. The xanthophyll cycle, in which the carotenoid pigment Violaxanthin is reversibly converted into zeaxanthin, is ubiquitous among green algae and plants and is necessary for the regulation of light harvesting, protection from oxidative stress and adaptation to different light conditions1,2. Violaxanthin De-Epoxidase (VDE) is the key enzyme responsible for zeaxanthin synthesis from Violaxanthin under excess light. Here we show that the Chlorophycean VDE (CVDE) gene from the model green alga Chlamydomonas reinhardtii encodes an atypical VDE. This protein is not homologous to the VDE found in plants and is instead related to a lycopene cyclase from photosynthetic bacteria3. Unlike the plant-type VDE that is located in the thylakoid lumen, the Chlamydomonas CVDE protein is located on the stromal side of the thylakoid membrane. Phylogenetic analysis suggests that CVDE evolved from an ancient De-Epoxidase that was present in the common ancestor of green algae and plants, providing evidence of unexpected diversity in photoprotection in the green lineage. Chlamydomonas reinhardtii possesses an atypical Violaxanthin De-Epoxidase, homologous to a bacterial enzyme rather than plant or algal enzymes with the same function. This illustrates an unexpected diversity of photoprotection mechanisms in the green lineage of photosynthetic organisms.

  • Chlamydomonas Xanthophyll Cycle Mutants ldentified by Video Imaging of Chlorophyll Fluorescence Quenching
    2013
    Co-Authors: Krishna K. Niyogi, Olle Bjorkman, Arthur R. Grossman
    Abstract:

    The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the ApH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from Violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital videoimaging system. In excessive light, the npql mutant is unable to convert Violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by Violaxanthin De-Epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and Violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, ApH-dependent nonphotochemical quenching in the npql mutant demonstrates that the formation of zeaxanthin and antheraxanthin via Violaxanthin De-Epoxidase activity is not required for all ApH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light

  • Ascorbate Deficiency Can Limit Violaxanthin De-Epoxidase Activity in Vivo
    Plant physiology, 2002
    Co-Authors: Patricia Müller-moulé, Patricia L. Conklin, Krishna K. Niyogi
    Abstract:

    As a response to high light, plants have evolved non-photochemical quenching (NPQ), mechanisms that lead to the dissipation of excess absorbed light energy as heat, thereby minimizing the formation of dangerous oxygen radicals. One component of NPQ is pH dependent and involves the formation of zeaxanthin from Violaxanthin. The enzyme responsible for the conversion of Violaxanthin to zeaxanthin is Violaxanthin De-Epoxidase, which is located in the thylakoid lumen, is activated by low pH, and has been shown to use ascorbate (vitamin C) as its reductant in vitro. To investigate the effect of low ascorbate levels on NPQ in vivo, we measured the induction of NPQ in a vitamin C-deficient mutant of Arabidopsis, vtc2-2 . During exposure to high light (1,500 μmol photons m −2 s −1 ), vtc2-2 plants initially grown in low light (150 μmol photons m −2 s −1 ) showed lower NPQ than the wild type, but the same quantum efficiency of photosystem II. Crosses between vtc2-2 and Arabidopsis ecotype Columbia established that the ascorbate deficiency cosegregated with the NPQ phenotype. The conversion of Violaxanthin to zeaxanthin induced by high light was slower in vtc2-2 , and this conversion showed saturation below the wild-type level. Both the NPQ and the pigment phenotype of the mutant could be rescued by feeding ascorbate to leaves, establishing a direct link between ascorbate, zeaxanthin, and NPQ. These experiments suggest that ascorbate availability can limit Violaxanthin De-Epoxidase activity in vivo, leading to a lower NPQ. The results also demonstrate the interconnectedness of NPQ and antioxidants, both important protection mechanisms in plants.

  • Photoprotection in a zeaxanthin- and lutein-deficient double mutant of Arabidopsis.
    Photosynthesis Research, 2001
    Co-Authors: Krishna K. Niyogi, Barry J. Pogson, Connie Shih, Wah Soon Chow, Dean Dellapenna, Olle Bjorkman
    Abstract:

    When light absorption by a plant exceeds its capacity for light utilization, photosynthetic light harvesting is rapidly downregulated by photoprotective thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). To address the involvement of specific xanthophyll pigments in NPQ, we have analyzed mutants affecting xanthophyll metabolism in Arabidopsis thaliana. An npq1 lut2 double mutant was constructed, which lacks both zeaxanthin and lutein due to defects in the Violaxanthin De-Epoxidase and lycopene ∈-cyclase genes. The npq1 lut2 strain had normal Photosystem II efficiency and nearly wild-type concentrations of functional Photosystem II reaction centers, but the rapidly reversible component of NPQ was completely inhibited. Despite the defects in xanthophyll composition and NPQ, the npq1 lut2 mutant exhibited a remarkable ability to tolerate high light.

  • photodamage of the photosynthetic apparatus and its dependence on the leaf developmental stage in the npq1 arabidopsis mutant deficient in the xanthophyll cycle enzyme Violaxanthin de epoxidase
    Plant Physiology, 2000
    Co-Authors: Michel Havaux, Jeanpaul Bonfils, Cornelius Lutz, Krishna K. Niyogi
    Abstract:

    The npq1 Arabidopsis mutant is deficient in the Violaxanthin De-Epoxidase enzyme that converts Violaxanthin to zeaxanthin in excess light (xanthophyll cycle). We have compared the behavior of mature leaves (ML) and developing leaves of the mutant and the wild type in various light environments. Thermoluminescence measurements indicated that high photon flux densities (>500 μmol m −2 s −1 ) promoted oxidative stress in the chloroplasts of npq1 ML, which was associated with a loss of chlorophyll and an inhibition of the photochemical activity. Illuminating leaf discs in the presence of eosin, a generator of singlet oxygen, brought about pronounced lipid peroxidation in npq1 ML but not in wild-type leaves. No such effects were seen in young leaves (YL) of npq1 , which were quite tolerant to strong light and eosin-induced singlet oxygen. Non-photochemical energy quenching was strongly inhibited in npq1 YL and ML and was not improved with high-light acclimation. Our results confirm that the xanthophyll cycle protects chloroplasts from photooxidation by a mechanism distinct from non-photochemical energy quenching and they reveal that the absence of xanthophyll cycle can be compensated by other protective mechanisms. npq1 YL were observed to accumulate considerable amounts of vitamin E during photoacclimation, suggesting that this lipophilic antioxidant could be involved in the high phototolerance of those leaves.

Robert C. Bugos - One of the best experts on this subject based on the ideXlab platform.

  • overexpression of Violaxanthin de epoxidase properties of c terminal deletions on activity and ph dependent lipid binding
    Planta, 2002
    Co-Authors: David A Hieber, Robert C. Bugos, Amy S. Verhoeven, Harry Y. Yamamoto
    Abstract:

    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 non-photochemical 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.

  • Violaxanthin De-Epoxidase: Properties of C-terminal deletions on activity, NPQ and pH-dependent lipid binding in tobacco
    Science Access, 2001
    Co-Authors: Andrew David Hieber, Robert C. Bugos, Amy S. Verhoeven, Harry Yamamoto
    Abstract:

    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. The full-length Arabidopsis VDE cDNA under either the single 35S cauliflower mosaic virus (CaMV) promoter or the double 35S CaMV promoter was used to transformed tobacco plants. Overexpression of VDE under control of the double 35S CaMV promoter increased VDE specific activity in the thylakoid lumen by 18-fold, relative to wild-type. These plants also demonstrated an increase in the initial rate of non-photochemical quenching (NPQ) consistent with increased rates of de-epoxidation. The glutamate-rich C-terminal region of VDE was subjected to analysis using C-terminal deletion mutants to understand the importance of the C-terminal domain in binding to the thylakoid membrane. Transformation of tobacco with two deletion mutants demonstrated that 71 C-terminal amino acids could be removed without affecting activity. In-vitro lipid-micelle binding experiments using these mutants identified a region of 12 amino acids that is potentially part of the membrane-binding domain. These transformed tobacco plants are the first reported example of plants with an increased level of Violaxanthin De-Epoxidase activity.

  • Plant lipocalins: Violaxanthin De-Epoxidase and zeaxanthin epoxidase.
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Arleen D. Hieber, Robert C. Bugos, Harry Y. Yamamoto
    Abstract:

    Abstract Violaxanthin De-Epoxidase and zeaxanthin epoxidase catalyze the interconversions between the carotenoids Violaxanthin, antheraxanthin and zeaxanthin in plants. These interconversions form the Violaxanthin or xanthophyll cycle that protects the photosynthetic system of plants against damage by excess light. These enzymes are the first reported lipocalin proteins identified from plants and are only the second examples of lipocalin proteins with enzymatic activity. This review summarizes the discovery and characterization of these two unique lipocalin enzymes and examines the possibility of other potential plant lipocalin proteins.

  • Antisense suppression of Violaxanthin De-Epoxidase in tobacco does not affect plant performance in controlled growth conditions.
    Photosynthesis research, 2000
    Co-Authors: Sue-hwei Chang, Robert C. Bugos, Wen-hao Sun, Harry Y. Yamamoto
    Abstract:

    Violaxanthin De-Epoxidase (VDE) catalyzes the de-epoxidation of Violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Tobacco was transformed with an antisense VDE construct under control of the cauliflower mosaic virus 35S promoter to determine the effect of reduced levels of VDE on plant growth. Screening of 40 independent transformants revealed 18 antisense lines with reduced levels of VDE activity with two in particular (TAS32 and TAS39) having greater than 95% reduction in VDE activity. Northern analysis demonstrated that these transformants had greatly suppressed levels of VDE mRNA. De-epoxidation of Violaxanthin was inhibited to such an extent that no zeaxanthin and only very low levels of antheraxanthin could be detected after exposure of leaves to high light (2000 μmol m−2 s−1 for 20 min) with no observable effect on levels of other carotenoids and chlorophyll. Non-photochemical quenching was greatly reduced in the antisense VDE tobacco, demonstrating that a significant level of the non-photochemical quenching in tobacco requires de-epoxidation of Violaxanthin. Although the antisense plants demonstrated a greatly impaired de-epoxidation of Violaxanthin, no effect on plant growth or photosynthetic rate was found when plants were grown at a photon flux density of 500 or 1000 μmol m−2 s−1 under controlled growth conditions as compared to wild-type tobacco.

  • Developmental Expression of Violaxanthin De-Epoxidase in Leaves of Tobacco Growing under High and Low Light
    Plant physiology, 1999
    Co-Authors: Robert C. Bugos, Sue-hwei Chang, Harry Y. Yamamoto
    Abstract:

    Violaxanthin De-Epoxidase (VDE) is a lumen-localized enzyme that catalyzes the de-epoxidation of Violaxanthin in the thylakoid membrane upon formation of a transthylakoid pH gradient. We investigated the developmental expression of VDE in leaves of mature tobacco (Nicotiana tabacum) plants grown under high-light conditions (in the field) and low-light conditions (in a growth chamber). The difference in light conditions was evident by the increased pool size (Violaxanthin + antheraxanthin + zeaxanthin, VAZ) throughout leaf development in field-grown plants. VDE activity based on chlorophyll or leaf area was low in the youngest leaves, with the levels increasing with increasing leaf age in both high- and low-light-grown plants. However, in high-light-grown plants, the younger leaves in early leaf expansion showed a more rapid increase in VDE activity and maintained higher levels of VDE transcript in more leaves, indicating that high light may induce greater levels of VDE. VDE transcript levels decreased substantially in leaves of mid-leaf expansion, while the levels of enzyme continued to increase, suggesting that the VDE enzyme does not turn over rapidly. The level of VDE changed in an inverse, nonlinear relationship with respect to the VAZ pool, suggesting that enzyme levels could be indirectly regulated by the VAZ pool.

Dariusz Latowski - One of the best experts on this subject based on the ideXlab platform.

  • amino sugars new inhibitors of zeaxanthin epoxidase a Violaxanthin cycle enzyme
    Journal of Plant Physiology, 2007
    Co-Authors: Dariusz Latowski, Kazimierz Strzalka, Agnieszka Katarzyna Banaś, Halina Gabryś
    Abstract:

    The effect of three sugars and their amino derivatives on Violaxanthin cycle enzymes activity was investigated in duckweed (Lemna trisulca), a model water-plant. No effect of sugars and amino sugars on Violaxanthin De-Epoxidase was observed independent of incubation time; however, epoxidation of zeaxanthin to Violaxanthin was inhibited. The minimum amino sugar concentrations causing maximum inhibition of zeaxanthin epoxidation have been estimated. Amino sugars but not sugars caused more than a 50% inhibition of zeaxanthin epoxidation in duckweed after a 24h incubation when applied at a concentration of 0.5%. Incubation with amino sugars under a 6d photoperiod enhanced the inhibitory effect. Zeaxanthin epoxidation was completely inhibited under such conditions, whereas only a minor inhibitory effect was observed in sugar treated plants. The strong amino sugar inhibition of zeaxanthin epoxidase activity represents additional evidence for the creation of an unstable carotenoid carbocation in the molecular mechanism of epoxidation.

  • Amino sugars – new inhibitors of zeaxanthin epoxidase, a Violaxanthin cycle enzyme
    Journal of plant physiology, 2006
    Co-Authors: Dariusz Latowski, Kazimierz Strzałka, Agnieszka Katarzyna Banaś, Halina Gabryś
    Abstract:

    The effect of three sugars and their amino derivatives on Violaxanthin cycle enzymes activity was investigated in duckweed (Lemna trisulca), a model water-plant. No effect of sugars and amino sugars on Violaxanthin De-Epoxidase was observed independent of incubation time; however, epoxidation of zeaxanthin to Violaxanthin was inhibited. The minimum amino sugar concentrations causing maximum inhibition of zeaxanthin epoxidation have been estimated. Amino sugars but not sugars caused more than a 50% inhibition of zeaxanthin epoxidation in duckweed after a 24h incubation when applied at a concentration of 0.5%. Incubation with amino sugars under a 6d photoperiod enhanced the inhibitory effect. Zeaxanthin epoxidation was completely inhibited under such conditions, whereas only a minor inhibitory effect was observed in sugar treated plants. The strong amino sugar inhibition of zeaxanthin epoxidase activity represents additional evidence for the creation of an unstable carotenoid carbocation in the molecular mechanism of epoxidation.

  • Inhibition of zeaxanthin epoxidase activity by cadmium ions in higher plants.
    Journal of inorganic biochemistry, 2005
    Co-Authors: Dariusz Latowski, Jerzy Kruk, Kazimierz Strzałka
    Abstract:

    The effect of cadmium and zinc ions on Violaxanthin cycle enzymes, Violaxanthin De-Epoxidase and zeaxanthin epoxidase, has been investigated on selected plant species, as well as in vitro. About 50% inhibition of zeaxanthin epoxidase by cadmium ions was found for duckweed (Lemna trisulca) and tomato (Lycopersicon esculentum) leaves but for apricot (Prunus armeniaca) leaves no cadmium inhibition of the epoxidation reaction was observed. The cadmium inhibition of zeaxanthin epoxidase in tomato was abolished by zinc ions. Zinc ions alone did not affect the activity of neither of the enzymes of the Violaxanthin cycle. This suggests that mechanism of cadmium inactivation of the enzyme relies on cadmium interaction with a cysteine residue of the protein, important for the enzyme activity. The target cysteine in tomato epoxidase could be the cysteine residue present in the most conservative part of the molecule which is not present in the apricot enzyme sequence. Neither stimulation nor inhibition of Violaxanthin De-Epoxidase by cadmium ions both in vivo and in vitro studies was detected. It confirms the proposed mechanism of zeaxanthin epoxidation inhibition by cadmium ions because the cysteine residue in the conservative motif of violaxathin De-Epoxidase is not present.

  • the xanthophyll cycle molecular mechanism and physiological significance
    Acta Physiologiae Plantarum, 2004
    Co-Authors: Joanna Grzyb, Dariusz Latowski, Kazimierz Strzalka
    Abstract:

    The light-dependent, cyclic changes of xanthophyll pigments: Violaxanthin, antheraxanthin and zeaxanthin, called the xanthophyll cycle, have been known for about fifty years. This process was characterised for higher plants, several fern and moss species and in some algal groups. Two enzymes, Violaxanthin De-Epoxidase (VDE) and zeaxanthin epoxidase (ZE), belonging to the lipocalin protein family, are engaged in the xanthophyll cycle. VDE requires for its activity ascorbic acid and reversed hexagonal structure formed by monogalactosyldiacylglycerol. ZE, postulated to be a flavoprotein, has not been purified yet and it is known from its gene sequence only. Zeaxanthin epoxidation is dependent on the reducing power of NADPH and presence of additional proteins.

  • Violaxanthin de epoxidase the xanthophyll cycle enzyme requires lipid inverted hexagonal structures for its activity
    Biochemistry, 2004
    Co-Authors: Dariusz Latowski, Hans-erik Åkerlund, Kazimierz Strzalka
    Abstract:

    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.