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Biomedical subjects

H Y Yamamoto

Publications and source records attributed to H Y Yamamoto.

At least 19 recordsLinked to original sources
PubMed2001

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

Tobacco (Nicotiana tabacum cv. Xanthi) transformed with an antisense cDNA construct of violaxanthin de-epoxidase (VDE) was examined for the effects of suppressed xanthophyll-cycle activity on photoinhibition, photosynthesis and growth under field conditions. De-epoxidation of violaxanthin and non-photochemical quenching were highly inhibited in antisense plants relative to vector-control and wild-type plants. However, no differences were observed between antisense and control plants in photosynthetic CO(2) uptake and maximum photochemical yield [(F(m)-F(o))/F(m)] measured at predawn or in actual photochemical yield [(F(m)'-F(s))/F(m)'] measured at midday. Moreover, growth rates of the plants were the same, as were the leaf area ratio, plant height and leaf number. Similarly, antisense plants did not exhibit greater susceptibility to photoinhibition than controls under field conditions. In contrast, when chloroplast protein (D1) synthesis was inhibited by lincomycin, antisense plants were more vulnerable to photoinhibition than wild-type plants. These results indicate that photoprotection under field conditions is not strictly dependent on the levels of the de-epoxidized xanthophylls, antheraxanthin and zeaxanthin.

Journal Article↗
PubMed2001

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

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

Journal Article↗
PubMed2001

Time-resolution of the antheraxanthin- and delta pH-dependent chlorophyll a fluorescence components associated with photosystem II energy dissipation in Mantoniella squamata.

The electronic excited-state behavior of photosystem II (PSII) in Mantoniella squamata, as influenced by the xanthophyll cycle and the transthylakoid pH gradient (delta pH), was examined in vivo. Mantoniella is distinguished from other photosynthetic organisms by two main features namely (1) a unique light-harvesting complex that serves both photosystems I (PSI) and II (PSII); and (2) a violaxanthin (V) cycle that undergoes only one de-epoxidation step in excess light to accumulate the monoepoxide antheraxanthin (A) as opposed to the epoxide-free zeaxanthin (Z). The cells were treated first with high light to induce the delta pH and A accumulation, followed by herbicide-induced closure of PSII traps and a chilling treatment, to sustain and stabilize the delta pH and nigericin-sensitive fluorescence level in the dark. De-epoxidation was controlled with subsaturating concentrations of dithiothreitol (DTT) and was 5-10 times more sensitive to DTT than higher plant thylakoids. The PSII energy dissipation involved two steps: (1) the pH activation of the xanthophyll binding site that was associated with a narrowing and slight attenuation of the main 2 ns (ns = 10(-9) s) fluorescence lifetime distribution; and (2) the concentration-dependent binding of A to the activated binding site yielding a second distribution centered around 0.9 ns. Consistent with the model of Gilmore et al. (1998) (Biochemistry 37, 13,582-13,593), the fractional intensity of the 0.9 ns component depended almost entirely on the A concentration and correlated linearly with the decrease of the steady-state chlorophyll alpha fluorescence intensity.

Carotenoids↗
PubMed2000

Plant lipocalins: violaxanthin de-epoxidase and zeaxanthin epoxidase.

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.

Amino Acid Sequence↗
PubMed2000

Antisense suppression of violaxanthin de-epoxidase in tobacco does not affect plant performance in controlled growth conditions.

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 mumol 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 mumol m(-2) s(-1) under controlled growth conditions as compared to wild-type tobacco.

Journal Article↗
PubMed1999

Developmental expression of violaxanthin de-epoxidase in leaves of tobacco growing under high and low light.

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.

Chlorophyll↗
PubMed1998

Xanthophyll cycle enzymes are members of the lipocalin family, the first identified from plants.

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 and Arabidopsis 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 beta-strands forming a barrel configuration. This is the first reported identification of lipocalin proteins in plants.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.

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.

Amino Acid Sequence↗
PubMed1996

Violaxanthin de-epoxidase.

Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photochemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy in the antennae. Previously, violaxanthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a novel lipid-affinity precipitation step with monogalactosyldiacylglyceride. The association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.

Amino Acid Sequence↗
PubMed1994

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

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

Carotenoids↗
PubMed1992

Dark induction of zeaxanthin-dependent nonphotochemical fluorescence quenching mediated by ATP.

Zeaxanthin-dependent nonphotochemical fluorescence quenching is a light-induced activity in plants that apparently protects against the potentially damaging effects of excess light. We report a dark-induced nonphotochemical quenching in thylakoids of Lactuca sativa L. cv. Romaine mediated by ATP. This effect is due to low lumen pH from hydrolysis-dependent proton pumping and hence required an active ATPase. The induction was optimal at 0.3 mM ATP, a physiological concentration, and occurred under conditions of little or no reverse electron flow. The properties of ATP-induced quenching were in all respects examined similar to light-induced quenching, including antimycin inhibition of quenching induction but not delta pH. We conclude that zeaxanthin-dependent quenching depends directly on lumen pH and that the role of light is indirect. Although it is known that zeaxanthin and low lumen pH are insufficient for quenching to occur, the results apparently exclude the redox state of an electron-transport carrier or formation of light-induced carotenoid triplets as a further requirement. We propose that a slow pH-dependent conformational change together with zeaxanthin cause static quenching in the pigment bed; possibly antimycin inhibits this change. Furthermore, we suggest from the ability of ATP to sustain quenching in the dark for extended periods that persistent or slowly reversible zeaxanthin quenching often observed in vivo may be due to sustained delta pH from ATP hydrolysis.

Adenosine Triphosphate↗
PubMed1992

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts.

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO(2)-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required DeltapH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and DeltapH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO(2) fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H(2)O(2). Ascorbate is essential for this process to occur.

Journal Article↗
PubMed1991

Zeaxanthin Formation and Energy-Dependent Fluorescence Quenching in Pea Chloroplasts under Artificially Mediated Linear and Cyclic Electron Transport.

Artificially mediated linear (methylviologen) and cyclic (phenazine methosulfate) electron transport induced zeaxanthin-dependent and independent (constitutive) nonphotochemical quenching in osmotically shocked chloroplasts of pea (Pisum sativum L. cv Oregon). Nonphotochemical quenching was quantitated as Stern-Volmer quenching (SV(N)) calculated as (F(m)/F'(m))-1 where F(m) is the fluorescence intensity with all PSII reaction centers closed in a nonenergized, dark-adapted state and F'(m) is the fluorescence intensity with all PSII reaction centers closed in an energized state. Reversal of quenching by nigericin and electron-transport inhibitors showed that both quenching types were energy-dependent SV(N). Under light-induced saturating DeltapH, constitutive-SV(N) reached steady-state in about 1 minute whereas zeaxanthin-SV(N) continued to develop for several minutes in parallel with the slow kinetics of violaxanthin deepoxidation. SV(N) above the constitutive level and relative zeaxanthin concentration showed high linear correlations at steady-state and during induction. Furthermore, F(o) quenching, also treated as Stern-Volmer quenching (SV(O)) and calculated as (F(o)/F'(o))-1, showed high correlation with zeaxanthin and consequently with SV(N) (F(o) and F'(o) are fluorescence intensities with all PSII reaction centers in nonenergized and energized states, respectively). These results support the view that zeaxanthin increases SV(N) above the constitutive level in a concentration-dependent manner and that zeaxanthin-dependent SV(N) occurs in the pigment bed. Preforming zeaxanthin increased the rate and extent of SV(N), indicating that slow events other than the amount of zeaxanthin also affect final zeaxanthin-SV(N) expression. The redox state of the primary electron acceptor of photosystem II did not appear to determine SV(N). Antimycin, when added while chloroplasts were in a dark-adapted or nonenergized state, inhibited both zeaxanthin-SV(N) and constitutive-SV(N) induced by linear and cyclic electron transport. These similarities, including possible constitutive F(o) quenching, suggest that zeaxanthin-dependent and constitutive SV(N) are mechanistically related.

Journal Article↗
PubMed1976

Light-induced De-epoxidation in Lettuce Chloroplasts: VI. De-epoxidation in Grana and in Stoma Lamellae.

Grana and stroma lamellae fractions prepared from illuminated chloroplasts (Lactuca sativa L. var. Manoa) by French press treatment contained less violaxanthin and more zeaxanthin than the corresponding fractions from dark controls. In both fractions, only part of the total violaxanthin was de-epoxidized under illumination, and the ratio of de-epoxidized and unchanged violaxanthin was similar. This not only shows that the de-epoxidation system is present in both grana and stroma thylakoids but also that violaxanthin is heterogeneous in both membranes. The presence and similarity of the de-epoxidation system in grana and stroma lamellae suggest that the function of the violaxanthin cycle is linked to photosynthetic activities which are common to both types of membranes.

Journal Article↗
PubMed1975

Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts. IV. The effects of electron-transport conditions on violaxanthin availability.

1. In isolated chloroplasts of Lactuca sativa var. Manoa, the size of the violaxanthin fraction which is available for de-epoxidation is not directly dependent on electron transport but rather related to the reduced level of some electron carrier between the photosystems. This is concluded from the effects of various electron-transport conditions on violaxanthin availability: Under conditions of electron transport through both photosystems, availability was saturated at a lower electron-transport rate with actinic light at 670 than at 700 nm. Under conditions of electron transport through Photosystem I, availability was smaller for linear electron flow from reduced N-methylphenazonium methosulfate via methylviologen to oxygen than from cyclic electron flow mediated by either N-methylphenazonium methosulfate or 2,6-dichlorophenolindophenol; in addition for linear flow from reduced N-methyphenazonium methosulfate via methylviologen to oxygen, availability increased with decreasing light intensity. 2. The postulated carrier whose reduced level is related to availability seems to be some carrier between plastoquinone and the primary acceptor of Photosystem II or plastoquinone itself. This conclusion follows from the fact that availability increased with increasing light intensity under conditions of electron flow through both photosystems and that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (greater than or equal to 1 mu M) had no effect on availability, whereas low levels of 3, 3-(3',4'-dichlorophenyl)-1,1-dimethylurea resulted in decreased availability (50 percent decrease at 1 mu M). Furthermore, availability in 3,3-(3',4'-dichlorophenyl)-1,1-dimethylurea-poisoned chloroplasts was fully restored by 2-methyl-1,4-naphtoquinone (menadione) which mediates cyclic electron flow through plastoquinone. 3. Violaxanthin availability was zero in the dark and increased in the light to maximum of 67 percent of the total violazanthin in chloroplasts. It is proposed that this variable violaxanthin availability reflects conformational changes on the internal surface of the thylakoid membrane which result in variable exposure of violaxanthin to the de-epoxidase. The fact that not all of the violaxanthin was available for de-epoxidation may indicate a heterogenous distribution of violaxanthin in the membrane.

Aerobiosis↗