Identification of key amino acids responsible for the distinct aggregation properties of microtubule-associated protein 2 and tau.

The carboxyl-terminal sequence of tau composes the framework for its intracellular inclusions that appear in diverse neurodegenerative disorders known as tauopathies. However, microtubule-associated protein 2 (MAP2), which contains a homologous carboxyl-terminal sequence of tau, is undetectable in the mature tau inclusions. The mechanisms underlying this phenomenon have remained largely unknown. Here, we show that tau and MAP2 have different aggregation properties: tau aggregates to form filaments but MAP2 remains to be granules. Exchanging (221) YKPV(224) of tau (0N3R) near the PHF6 motif for (340) TKKI(343) of MAP2c profoundly changed aggregation properties, suggesting that the YKPV motif is important for filament formation, whereas the TKKI motif is for granule formation. Thus, these minimal sequences may determine the different fates of tau and MAP2 in the formation of inclusions in tauopathies. Tau and microtubule-associated protein 2 (MAP2) are homologous microtubule-associated proteins in neurons. So far, it is largely unknown why tau but not MAP2 is selectively involved in the filamentous inclusions (neurofibrillary tangles, NFT) formation in tauopathies, including Alzheimer's disease. In this study, we found that the difference of only two amino acids in tau and MAP2 sequences may determine their different fates in tauopathies. These results may lead to the elucidation of tau deregulation in pathological conditions.

Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering.

SUMMARY: The natural pigment astaxanthin has attracted much attention because of its beneficial effects on human health, despite its expensive market price. In order to produce astaxanthin, transgenic plants have so far been generated through conventional genetic engineering of Agrobacterium-mediated gene transfer. The results of trials have revealed that the method is far from practicable because of low yields, i.e. instead of astaxanthin, large quantities of the astaxanthin intermediates, including ketocarotenoids, accumulated in the transgenic plants. In the present study, we have overcome this problem, and have succeeded in producing more than 0.5% (dry weight) astaxanthin (more than 70% of total caroteniods) in tobacco leaves, which turns their green color to reddish brown, by expressing both genes encoding CrtW (beta-carotene ketolase) and CrtZ (beta-carotene hydroxylase) from a marine bacterium Brevundimonas sp., strain SD212, in the chloroplasts. Moreover, the total carotenoid content in the transplastomic tobacco plants was 2.1-fold higher than that of wild-type tobacco. The tobacco transformants also synthesized a novel carotenoid 4-ketoantheraxanthin. There was no significant difference in the size of the aerial part of the plant between the transformants and wild-type plants at the final stage of their growth. The photosynthesis rate of the transformants was also found to be similar to that of wild-type plants under ambient CO2 concentrations of 1500 micromol photons m(-2) s(-1) light intensity.

Imbalanced Expression of Tau and Tubulin Induces Neuronal Dysfunction in C. elegans Models of Tauopathy.

Tauopathy is a type of dementia defined by the accumulation of filamentous tau inclusions in neural cells. Most types of dementia in the elderly, including Alzheimer's disease, are tauopathies. Although it is believed that tau protein abnormalities and/or the loss of its functions results in neurodegeneration and dementia, the mechanism of tauopathy remains obscure. Loss of microtubules and/or tubulin is a known consequence of tau accumulating in neurons in Alzheimer's disease. In other words, there is an excess level of tau relative to tubulin in tauopathy neurons. To test whether this imbalance of tau and tubulin expression results in the neurotoxicity of tau, we developed several transgenic C. elegans lines that express human tau at various levels in pan-neurons. These worms showed behavioral abnormalities in a tau expression-dependent manner. The knockdown of a tubulin-specific chaperon, or a subset of tubulin, led to enhanced tau toxicity even in low-expressing tau-transgenic worms that showed no abnormal behaviors. In addition, the suppression of tau expression in tubulin knockdown worms rescued neuronal dysfunction. Thus, not only the overexpression of tau but also a reduction in tubulin can trigger the neurotoxicity of tau. Tau expressed in worms was also highly phosphorylated and largely bound to tubulin dimers rather than microtubules. Relative amount of tubulin-unbound tau was increased in high-expressing tau-transgenic worms showing tau toxicity. We further demonstrated that tau aggregation was inhibited by co-incubation of purified tubulin in vitro, meaning sufficient amounts of tubulin can protect against the formation of tau inclusions. These results suggest that the expression ratio of tau to tubulin may be a determinant of the tauopathy cascade.

Curcumin improves tau-induced neuronal dysfunction of nematodes.

Tau is a key protein in the pathogenesis of various neurodegenerative diseases, which are categorized as tauopathies. Because the extent of tau pathologies is closely linked to that of neuronal loss and the clinical symptoms in Alzheimer's disease, anti-tau therapeutics, if any, could be beneficial to a broad spectrum of tauopathies. To learn more about tauopathy, we developed a novel transgenic nematode (Caenorhabditis elegans) model that expresses either wild-type or R406W tau in all the neurons. The wild-type tau-expressing worms exhibited uncoordinated movement (Unc) and neuritic abnormalities. Tau accumulated in abnormal neurites that lost microtubules. Similar abnormalities were found in the worms that expressed low levels of R406W-tau but were not in those expressing comparative levels of wild-type tau. Biochemical studies revealed that tau is aberrantly phosphorylated but forms no detergent-insoluble aggregates. Drug screening performed in these worms identified curcumin, a major phytochemical compound in turmeric, as a compound that reduces not only Unc but also the neuritic abnormalities in both wild-type and R406W tau-expressing worms. Our observations suggest that microtubule stabilization mediates the antitoxicity effect of curcumin. Curcumin is also effective in the worms expressing tau fragment, although it does not prevent the formation of tau-fragment dimers. These data indicate that curcumin improves the tau-induced neuronal dysfunction that is independent of insoluble aggregates of tau.

Deactivation of aquaporins decreases internal conductance to CO2 diffusion in tobacco leaves grown under long-term drought.

We compared the diffusion conductance to CO2 from the intercellular air space to the chloroplasts (internal conductance (g i)) between tobacco leaves acclimated to long-term drought (drought-acclimated (DA)) and those grown under sufficient irrigation (well-watered (WW)), and analysed the changes in g i in relation to the leaf anatomical characteristics and a possible CO2 transporter, aquaporin. The g i, which was estimated by combined analyses of CO2 gas exchange with chlorophyll fluorescence, in the DA plants was approximately half of that in the WW plants. The mesophyll and chloroplast surface areas exposing the intercellular air space, which potentially affect g i, were not significantly different between the WW and DA plants. The amounts of plasma membrane aquaporins (PIP), immunochemically determined using radish PIP antibodies, were unrelated to g i. After treatment with HgCl2, an aquaporin inhibitor, the water permeability of the leaf tissues (measured as the weight loss of fully-turgid leaf disks without the abaxial epidermis in 1 m sorbitol) in WW plants decreased with an increase in HgCl2 concentration. The g i in the WW plants decreased to similar levels to the DA plants when the detached leaflets were fed with 0.5 mm HgCl2. In contrast, both water permeability and g i were insensitive to HgCl2 treatments in DA plants. These results suggest that deactivation of aquaporins is responsible for the significant reduction in g i observed in plants growing under long-term drought.

Photoinactivation of ascorbate peroxidase in isolated tobacco chloroplasts: Galdieria partita APX maintains the electron flux through the water-water cycle in transplastomic tobacco plants.

We evaluated the H2O2-scavenging activity of the water-water cycle (WWC) in illuminated intact chloroplasts isolated from tobacco leaves. Illumination under conditions that limited photosynthesis [red light (>640 nm), 250 micromol photons m(-2) s(-1) in the absence of HCO3-] caused chloroplasts to take up O2 and accumulate H2O2. Concomitant with the O2 uptake, both ascorbate peroxidase (APX) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) lost their activities. However, superoxide dismutase (SOD), monodehydroascorbate radical reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) activities remained unaffected. The extent to which the photosynthetic linear electron flow decreased was small compared with the decline in APX activity. Therefore, the loss of APX activity lowered the electron flux through the WWC, as evidenced by a decrease in relative electron flux through PSII [Phi(PSII)xPFD]. To verify these interpretations, we created a transplastomic tobacco line in which an H2O2-insensitive APX from the red alga, Galdieria partita, was overproduced in the chloroplasts. In intact transplastomic chloroplasts which were illuminated under conditions that limited photosynthesis, neither O2 uptake nor H2O2 accumulation occurred. Furthermore, the electron flux through the WWC and the activity of GAPDH were maintained. The present work is the first report of APX inactivation by endogenous H2O2 in intact chloroplasts.

Ferredoxin limits cyclic electron flow around PSI (CEF-PSI) in higher plants--stimulation of CEF-PSI enhances non-photochemical quenching of Chl fluorescence in transplastomic tobacco.

We tested the hypothesis that ferredoxin (Fd) limits the activity of cyclic electron flow around PSI (CEF-PSI) in vivo and that the relief of this limitation promotes the non-photochemical quenching (NPQ) of Chl fluorescence. In transplastomic tobacco (Nicotiana tabacum cv Xanthi) expressing Fd from Arabidopsis (Arabidopsis thaliana) in its chloroplasts, the minimum yield (F(o)) of Chl fluorescence was higher than in the wild type. F(o) was suppressed to the wild-type level upon illumination with far-red light, implying that the transfer of electrons by Fd-quinone oxidoreductase (FQR) from the chloroplast stroma to plastoquinone was enhanced in transplastomic plants. The activity of CEF-PSI became higher in transplastomic than in wild-type plants under conditions limiting photosynthetic linear electron flow. Similarly, the NPQ of Chl fluorescence was enhanced in transplastomic plants. On the other hand, pool sizes of the pigments of the xanthophyll cycle and the amounts of PsbS protein were the same in all plants. All these results supported the hypothesis strongly. We conclude that breeding plants with an NPQ of Chl fluorescence increased by an enhancement of CEF-PSI activity might lead to improved tolerance for abiotic stresses, particularly under conditions of low light use efficiency.

CO2 response of cyclic electron flow around PSI (CEF-PSI) in tobacco leaves--relative electron fluxes through PSI and PSII determine the magnitude of non-photochemical quenching (NPQ) of Chl fluorescence.

We hypothesized that cyclic electron flow around photosystem I (CEF-PSI) participates in the induction of non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence when the rate of photosynthetic linear electron flow (LEF) is electron-acceptor limited. To test this hypothesis, the relationships among photosynthesis rate, electron fluxes through both PSI and PSII [Je(PSI) and Je(PSII)] and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants at several light intensities and partial pressures of ambient CO2 (Ca). At low light intensities, decreasing Ca lowered the photosynthesis rate, but Je(PSI) and Je(PSII) remained constant. Je(PSI) was larger than Je(PSII), indicating the existence of CEF-PSI. Increasing the light intensity enhanced photosynthesis and both Je(PSI) and Je (PSII). Je(PSI)/Je(PSII) also increased at high light and at high light and low Ca combined, showing a strong, positive relationship with NPQ of Chl fluorescence. These results indicated that CEF-PSI contributed to the dissipation of photon energy in excess of that consumed by photosynthesis by driving NPQ of Chl fluorescence. The main physiological function of CEF-PSI in photosynthesis of higher plants is discussed.

Effects of light intensity on cyclic electron flow around PSI and its relationship to non-photochemical quenching of Chl fluorescence in tobacco leaves.

We tested the hypothesis that plants grown under high light intensity (HL-plants) had a large activity of cyclic electron flow around PSI (CEF-PSI) compared with plants grown under low light (LL-plants). To evaluate the activity of CEF-PSI, the relationships between photosynthesis rate, quantum yields of both PSII and PSI, and Chl fluorescence parameters were analyzed simultaneously in intact leaves of tobacco plants which had been grown under different light intensities (150 and 1,100 micromol photons m(-2) s(-1), respectively) and with different amounts of nutrients supplied. HL-plants showed a larger value of non-photochemical quenching (NPQ) of Chl fluorescence at the limited activity of photosynthetic linear electron flow. Furthermore, HL-plants had a larger activity of CEF-PSI than LL-plants. These results suggested that HL-plants dissipated the excess photon energy through NPQ by enhancing the ability of CEF-PSI to induce acidification of the thylakoid lumen.

Enhancement of cyclic electron flow around PSI at high light and its contribution to the induction of non-photochemical quenching of chl fluorescence in intact leaves of tobacco plants.

Non-photochemical quenching (NPQ) of Chl fluorescence is a mechanism for dissipating excess photon energy and is dependent on the formation of a DeltapH across the thylakoid membranes. The role of cyclic electron flow around photosystem I (PSI) (CEF-PSI) in the formation of this DeltapH was elucidated by studying the relationships between O2-evolution rate [V(O2)], quantum yield of both PSII and PSI [Phi(PSII) and Phi(PSI)], and Chl fluorescence parameters measured simultaneously in intact leaves of tobacco plants in CO2-saturated air. Although increases in light intensity raised V(O2) and the relative electron fluxes through both PSII and PSI [Phi(PSII) x PFD and Phi(PSI) x PFD] only Phi(PSI) x PFD continued to increase after V(O2) and Phi(PSII) x PFD became light saturated. These results revealed the activity of an electron transport reaction in PSI not related to photosynthetic linear electron flow (LEF), namely CEF-PSI. NPQ of Chl fluorescence drastically increased after Phi(PSII) x PFD became light saturated and the values of NPQ correlated positively with the relative activity of CEF-PSI. At low temperatures, the light-saturation point of Phi(PSII) x PFD was lower than that of Phi(PSI) x PFD and NPQ was high. On the other hand, at high temperatures, the light-dependence curves of Phi(PSII) x PFD and Phi(PSI) x PFD corresponded completely and NPQ was not induced. These results indicate that limitation of LEF induced CEF-PSI, which, in turn, helped to dissipate excess photon energy by driving NPQ of Chl fluorescence.