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CO2 responsive CCT protein (CRCT) is a positive regulator of starch synthesis related genes such as ADP-glucose pyrophosphorylase large subunit 1 and starch branching enzyme I particularly in the leaf sheath of rice (Oryza sativa L.). The promoter GUS analysis revealed that CRCT expressed exclusively in the vascular bundle, whereas starch synthesis related genes were expressed in different sites such as mesophyll cell and starch storage parenchyma cell. However, the chromatin immunoprecipitation (ChIP) using a FLAG-CRCT overexpression line and subsequent qPCR analyses showed that the 5’-flanking regions of these starch synthesis-related genes tended to be enriched by ChIP, suggesting that CRCT can bind to the promoter regions of these genes. The monomer of CRCT is 34.2 kDa, however CRCT was detected at 270 kDa via gel filtration chromatography, suggesting that CRCT forms a complex in vivo. Immunoprecipitation and subsequent MS analysis pulled down several 14-3-3-like proteins. A yeast two-hybrid analysis and bimolecular fluorescence complementation assays confirmed the interaction between CRCT and 14-3-3-like proteins. Although there is an inconsistency in the place of expression, this study provide important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

One of the characteristic aspects of odour sensing in humans is the activation of olfactory receptors in a slightly different manner in response to different enantiomers. Here, we focused on whether plants showed enantiomer-specific response similar to that in humans. We exposed Arabidopsis seedlings to methanol (control) and (+)- or (−)-borneol, and found that only (+)-borneol reduced the root length. Furthermore, the root-tip width was more increased upon (+)-borneol exposure than upon (−)-borneol exposure. In addition, root-hair formation was observed near the root tip in response to (+)-borneol. Auxin signalling was strongly reduced in the root tip following exposure to (+)-borneol, but was detected following exposure to (−)-borneol and methanol. Similarly, in the root tip, the activity of cyclin B1:1 was detected on exposure to (−)-borneol and methanol, but not on exposure to (+)-borneol, indicating that (+)-borneol inhibits the meristematic activity in the root. These results partially explain the (+)-borneol-specific reduction in the root length of Arabidopsis. Our results indicate the presence of a sensing system specific for (+)-borneol in Arabidopsis.

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Green leaf volatiles (GLVs), which include C6 aldehydes, alcohols, and their esters, are emitted by damaged plants and are, therefore, thought to be involved in stress responses. However, the effects of GLVs on gene expression are not fully understood. Thus, the aim of the present study was to analyze the early transcriptional responses of Arabidopsis to the major GLVs—(Z)-3-hexenal, (Z)-3-hexenol, (E)-2-hexenal, and (Z)-3-hexenyl acetate—using comprehensive microarray gene expression analysis. All of the GLVs induced changes in gene expression, and (Z)-3-hexenal, (Z)-3-hexenol, and (Z)-3-hexenyl acetate commonly triggered the expression of defense-related genes, whereas (E)-2-hexenal mainly induced genes responsible for responding to abiotic stress, such as heat and oxidative stress. These results suggest that GLVs can function as airborne infochemicals that regulate the rapid expression of defense response-related genes and that GLVs might play a physiological role as self-made damage-associated molecular patterns (DAMPs) in damaged leaves.

Oxidative stimuli to living cells results in the formation of lipid peroxides, from which various aldehydes and ketones (oxylipin carbonyls) are inevitably produced. Among the oxylipin carbonyls, those with an α,β-unsaturated bond are designated as reactive carbonyl species (RCS) because they have high electrophilicity and biological activity. Plants have arrays of dehydrogenases and reductases to metabolize a variety of RCS that occur in the cells, but these enzymes are not efficient to scavenge the most toxic RCS (i.e., acrolein) because they have only low affinity. Two glutathione transferase (GST) isozymes belonging to the plant-specific Tau class were recently observed to scavenge acrolein with KM values at a submillimolar level. This suggests that GST could also be involved in the defense system against RCS. We tested the activities of 23 Tau isozymes of Arabidopsis thaliana for five types of RCS, and the results revealed that 11 isozymes recognized either acrolein or 4-hydroxy-(E)-2-nonenal or both as a substrate(s). Such RCS-scavenging activities indicate the potential contribution of GST to RCS scavenging in plants, and they may account for the stress tolerance conferred by several Tau isozymes. RCS are therefore a strong candidate for endogenous substrates of plant GSTs.

The gas exchange of the upper fully expanded leaf of the root parasite Striga hermonthica and of its host Sorghum bicolor was measured under wet and dry conditions to identify the mechanisms of the devastating effects of the parasite on its hosts under drought. The short-term water stress severely reduced photosynthetic rate in infected sorghum, but less in S. hermonthica. Soil water stress did not affect leaf respiration rate in either S. hermonthica or infected sorghum. This suggests that under dry conditions both infected sorghum and S. hermonthica decreased autotrophic carbon gain. The transpiration rate of S. hermonthica, a major driving force for assimilate uptake from the host, was higher and less affected by water stress than that of infected sorghum. Stomatal density on the abaxial surfaces of the leaves was higher in S. hermonthica than in sorghum. Both S. hermonthica infection and water stress decreased stomatal conductance of the sorghum leaves. S. hermonthica, irrespective of soil water status, had greater stomatal aperture on the adaxial and abaxial surfaces of its leaves than infected sorghum. These results indicate that the higher transpiration rate of S. hermonthica even under water stress, achieved through higher stomatal density on the abaxial surfaces of the leaves and greater stomatal aperture on both surfaces of the leaves, may induce the maintenance of water and solute transfers from the host to the parasite leading to severe damage to the host under drought.

The green odor of plants is characterized by green leaf volatiles (GLVs) composed of C6 compounds. GLVs are biosynthesized from polyunsaturated fatty acids in thylakoid membranes by a series of enzymes. A representative member of GLVs (E)-2-hexenal, known as the leaf aldehyde, has been assumed to be produced by isomerization from (Z)-3-hexenal in the biosynthesis pathway; however, the enzyme has not yet been identified. In this study, we purified the (Z)-3:(E)-2-hexenal isomerase (HI) from paprika fruits and showed that various plant species have homologous HIs. Purified HI is a homotrimeric protein of 110 kDa composed of 35-kDa subunits and shows high activity at acidic and neutral pH values. Phylogenetic analysis showed that HIs belong to the cupin superfamily, and at least three catalytic amino acids (His, Lys, Tyr) are conserved in HIs of various plant species. Enzymatic isomerization of (Z)-3-hexenal in the presence of deuterium oxide resulted in the introduction of deuterium at the C4 position of (E)-2-hexenal, and a suicide substrate 3-hexyn-1-al inhibited HI irreversibly, suggesting that the catalytic mode of HI is a keto-enol tautomerism reaction mode mediated by a catalytic His residue. The gene expression of HIs in Solanaceae plants was enhanced in specific developmental stages and by wounding treatment. Transgenic tomato plants overexpressing paprika HI accumulated (E)-2-hexenal in contrast to wild-type tomato plants mainly accumulating (Z)-3-hexenal, suggesting that HI plays a key role in the production of (E)-2-hexenal in planta.

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m-2 s-1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI-LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.

Under a moderately heat-stressed condition, the photosystems of higher plants are damaged in the dark more easily than they are in the presence of light. To obtain a better understanding of this heat-derived damage mechanism that occurs in the dark, we focused on the involvement of the light-independent electron flow that occurs at 40 °C during the damage. In various plant species, the maximal photochemical quantum yield of photosystem (PS) II (Fv/Fm) decreased as a result of heat treatment in the dark. In the case of wheat, the most sensitive plant species tested, both Fv/Fm and oxygen evolution rapidly decreased by heat treatment at 40 °C for 30 min in the dark. In the damage, specific degradation of D1 protein was involved, as shown by immunochemical analysis of major proteins in the photosystem. Because light canceled the damage to PSII, the light-driven electron flow may play a protective role against PSII damage without light. Light-independent incorporation of reducing power from stroma was enhanced at 40 °C but not below 35 °C. Arabidopsis mutants that have a deficit of enzymes which mediate the incorporation of stromal reducing power into thylakoid membranes were tolerant against heat treatment at 40 °C in the dark, suggesting that the reduction of the plastoquinone pool may be involved in the damage. In conclusion, the enhanced introduction of reducing power from stroma into thylakoid membranes that occurs around 40 °C causes over-reduction of plastoquinone, resulting in the damage to D1 protein under heat stress without linear electron flow.

Lipid peroxide-derived reactive carbonyls (RCs) can cause serious damage to plant functions. A chloroplastic NADPH-dependent alkenal/one oxidoreductase (AOR) detoxifies RCs, but its physiological significance remains unknown. In this study, we investigated the biological impacts of AOR using an AOR-knock out Arabidopsis line (aor). Methyl viologen treatment, mainly to enhance photosystem (PS) I-originated reactive oxygen species (ROS) production, caused more severe damage to aor than wild type (Col-0). In contrast, the high light treatment used to enhance PSII-originated ROS production resulted in no difference in PSII damage between Col-0 and aor. In conclusion, AOR can contribute to detoxify stromal RCs produced under oxidative stress.

Reactive carbonyls, especially α,β-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH(2) = CHCHO) as a model α,β-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an α,β-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced α,β-unsaturated ketones rather than α,β-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as α,β-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.

Previously we observed that the oxygen-evolving complex 33 kDa protein (OEC33) which stabilizes the Mn cluster in photosystem II (PSII), was modified with malondialdehyde (MDA), an end-product of peroxidized polyunsaturated fatty acids, and the modification increased in heat-stressed plants (Yamauchi et al. 2008). In this study, we examined whether the modification of OEC33 with MDA affects its binding to the PSII complex and causes inactivation of the oxygen-evolving complex. Purified OEC33 and PSII membranes that had been removed of extrinsic proteins of the oxygen-evolving complex (PSIIOEE) of spinach (Spinacia oleracea) were separately treated with MDA. The binding was diminished when both OEC33 and PSIIOEE were modified, but when only OEC33 or PSIIOEE was treated, the binding was not impaired. In the experiment using thylakoid membranes, release of OEC33 from PSII and corresponding loss of oxygen-evolving activity were observed when thylakoid membranes were treated with MDA at 40 degrees C but not at 25 degrees C. In spinach leaves treated at 40 degrees C under light, maximal efficiency of PSII photochemistry (F(v)/F(m) ratio of chlorophyll fluorescence) and oxygen-evolving activity decreased. Simultaneously, MDA contents in heat-stressed leaves increased, and OEC33 and PSII core proteins including 47 and 43 kDa chlorophyll-binding proteins were modified with MDA. In contrast, these changes were to a lesser extent at 40 degrees C in the dark. These results suggest that MDA modification of PSII proteins causes release of OEC33 from PSII and it is promoted in heat and oxidative conditions.

When polyunsaturated fatty acids (PUFAs) in biomembrane are peroxidized, a great diversity of aldehydes is formed, and some of which are highly reactive. Thus they are thought to have biological impacts in stressed plants; however, the detailed mechanism of generation and biochemical effects are unknown. In this study, we show that chloroplasts are major organelles in which malondialdehyde (MDA) generated from peroxidized linolenic acid modifies proteins in heat-stressed plants. First, to clarify the biochemical process of MDA generation from PUFAs and its attachment to proteins, we carried out in vitro experiments using model proteins (BSA and Rubisco) and methylesters of C18 PUFAs that are major components of plant biomembrane. Protein modification was detected by Western blotting using monoclonal antibodies that recognize MDA binding to proteins. Results showed that peroxidation of linolenic acid methylester by reactive oxygen species was essential for protein modification by MDA, and the MDA modification was highly dependent on temperature, leading to a loss of Rubisco activity. When isolated spinach thylakoid membrane was peroxidized at 37 degrees C, oxygen-evolving complex 33kDa protein (OEC33) was modified by MDA. These model experiments suggest that protein modification by MDA preferentially occurs under higher temperatures and oxidative conditions, thus we examined protein modification in heat-stressed plants. Spinach plants were heat-stressed at 40 degrees C under illumination, and modification of OEC33 protein by MDA was detected. In heat-stressed Arabidopsis plants, light-harvesting complex protein was modified by MDA under illumination. This modification was not observed in linolenic acid-deficient mutants (fad3fad7fad8 triple mutant), suggesting that linolenic acid is a major source of protein modification by MDA in heat-stressed plants.

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5'- and 3'-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17-1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970-20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293-51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.

We used suppression subtractive hybridization (SSH) to identify submergence-induced genes from the submergence-tolerant rice cultivar, FR13A. These genes putatively encode four proteins including a cation transport protein ( OsCTP ), monogalactosyl–diacylglycerol synthase, a cold-induced protein and glutathione synthetase. The full-length gene of one upregulated clone, OsCTP was generated by rapid amplification of cDNA ends. The gene comprises 741 bp with an open reading frame of 414 bp (nt 51–464) that encodes a putative protein of 137 amino acids. The theoretical isoelectric point of the putative protein is 5.35 and the molecular weight is 15.2 kDa. Six exons and five introns of the OsCTP gene correspond to 3582–2296 bp of accession number AAAA01013244 from the rice genome. OsCTP expression is enhanced by submergence as well as stress induced by abscisic acid, salt or drought. OsCTP might encode a novel cation transport protein similar to Escherichia coli ChaC and may be associated with a general defensive response to various environmental stresses.

Li 3−2 x M x InBr 6 (M=Mg, Ca, Sr, Ba; x =0–1.0) was synthesized and the cation substitution effect was investigated by means of X-ray diffraction, 7 Li and 115 In nuclear magnetic resonance (NMR), and AC conductivity measurements. In Li 3−2 x Mg x InBr 6 , the lattice constants a and c increased with an increase of x , but b and β did not show significant changes. In the high-temperature phase for Li 3−2 x M x InBr 6 (M=Mg, Ca, Ba), the conductivity decreased with x , accompanied by a broadening of the 115 In NMR spectra. In Li 3−2 x Sr x InBr 6 ( x ≤0.7), the conductivity of the high-temperature phase also decreased with an increase of x , but LiSrInBr 6 showed almost the same conductivity to Li 3 InBr 6 . On the other hand, the conductivity of the low-temperature phase in Li 3−2 x Mg x InBr 6 ( x ≤0.4) increased with x , associated with a narrowing of the 7 Li NMR spectra.

A chimeric gene consisting of the coding sequence for cytosolic Cu/Zn-superoxide dismutases (SOD) from Oryza sativa fused to the chloroplast transit sequence from Arabidopsis thaliana glutathione reductase was used for generating transgenic tobacco plants. This construct was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2. Tobacco leaf discs were infected with Agrobacterium and cultured on kanamycin selective medium. The integration and expression of Cu/Zn-SOD in tobacco genome was confirmed by Southern dot blot hybridization and SOD activity staining, respectively. Several transgenic lines were obtained and screened for SOD activity. The transgenic lines showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulfite. Similarly, when net photosynthesis was measured, the first generation of the transgenic lines showed enhanced tolerance to salt, water, and PEG stresses, over the wild type. These results suggested that the overexpressed Cu/Zn-SOD enhances the chloroplast antioxidant system.

Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25 degrees C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5-0.7 mol AsA incorporated (mol Rubisco)(-1) d(-1)). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, N( epsilon )-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7-8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.

The mechanism of flash flood tolerance was investigated by using a flash flood-intolerant rice cultivar (Oryza sativa), IR42 and a tolerant one, FR13A. The photosynthetic rate of the rice seedlings from both types of cultivars decreased during submergence, whereas the photosynthetic rate of FR13A remained higher than that of IR42. The decrease in photosynthesis in IR42 was partially due to a higher inhibition of the activities of ribulose-1,5-bisphosphate carboxylase, fructose-1,6-bisphosphatase, and glyceraldehyde-3-phosphate dehydrogenase, than in FR13A. After submergence receded (hereafter referred as “desubmergence”), both types of submerged rice cultivars experienced oxidative damage. However the oxidation of the lipids and proteins in FR13A was maintained at a lower level than that in IR42 after desubmergence. On the other hand, the superoxide dismutase activity in FR13A increased more than that in IR42 after desubmergence. Moreover, the content of total ascorbate (reduced and oxidized asco...

Li3-2x Mg InBr6 (x= 0.02 - 0.4) was synthesized, and the cation substitution effect on the conductivity was investigated by means of 7Li and 115In NMR, and X-ray diffraction.With increasing x the lattice constants a and c increased, but b and β did not show significant changes. The conductivity of the low temperature phase increased with x, associated with a narrowing of the 7Li NMR spectra. In the high temperature superionic phase, on the other hand, the conductivity decreased with x accompanied by a broadening of the 115In NMR spectra

When flash flood intolerant rice cultivars are submerged, they show greater morphological changes such as elongation and chlorosis than tolerant cultivars. These morphological responses are caused by ethylene produced during submergence, however, a visible damage of intolerant cultivars is markedly developing after desubmergence rather than during submergence, which is probably due to oxidative damage. We studied the effect of ethylene produced during submergence on antioxidant content and oxidative damage after desubmergence. When rice (Oryza sativa) was submerged for 8 days, both tolerant cultivar (BKNFR) and intolerant cultivars (Mashuri and IR42) showed a decrease in ascorbate concentration during submergence. After 3 days of desubmergence, the tolerant cultivar showed a rapid recovery of total ascorbate and ascorbic acid, whereas intolerant cultivars showed a slow recovery of them, an increase in malondialdehyde formation, and low survival rate (about 30%). However, applying 200 mg l−1 of AgNO3 as an ethylene antagonist to intolerant cultivars suppressed the decrease in ascorbate and the increase in malondialdehyde formation after desubmergence, and improved survival rate to about 60%. Ascorbic acid supply to leaf discs from submerged IR42 suppressed increase in malondialdehyde formation by incubation under the light for 24 h. In addition, strong negative correlations were observed between malondialdehyde formation with ascorbate concentration (r=−0.93) and with percentage of survival (r=−0.98). Our results indicate that the accumulated ethylene during submergence adversely affected antioxidant mechanism in intolerant rice cultivars after desubmergence, and ascorbic acid was an important antioxidant in vivo for the recovery of submerged rice seedlings.

In cucumber ( Cucumis sativus L.) leaves at different ontogenic stages, a differential appearance of three major endopeptidases was observed by employing activity staining using gelatin as a substrate. On the basis of this observation, we discussed their physiological roles in senescing leaves. The most active endopeptidase in young mature leaves was a glutamyl endopeptidase with a pI of 4.5. It might be involved in active protein catabolism in young leaves because its activity became maximal just after the leaf had fully expanded and when protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) contents rapidly decreased. An endopeptidase with a pI of 4.3 was not observed in young leaves, however, it was highly active in senescing leaves. Interestingly, its activity in cotyledons was eliminated when the upper metabolically active leaves were removed. This implies that the appearance of this enzyme is regulated by the presence of sink tissues, and it is involved in the degradation of protein in senescing leaves facilitating N transfer to upper developing leaves. A trypsin-like endopeptidase with a pI of 5.0 showed relatively constant activity during the whole period. This endopeptidase has been shown to be inhibited by arginine, guanidino compounds and Mg 2+ , therefore, it might exist constitutively and its activity might be regulated mainly at a post-translational level responding to nutrient and environmental conditions.

Photosystems of higher plants alleviate heat-induced damage in the presence of light under moderate stressed conditions, however, in the absence of light (i.e., in the dark), the same plants are damaged more easily. (Yamauchi and Kimura, 2011) We demonstrate that regulating photochemical energy transfer in heat-treated wheat at 40 °C with light contributed to heat tolerance of the photosystem. Chlorophyll fluorescence analysis using heat-stressed wheat seedlings in light showed increased non-photochemical quenching (NPQ) of chlorophyll fluorescence, which was due to thermal dissipation that was increased by state 1 to state 2 transition. Transmission electron microscopy revealed structural changes in thylakoid membranes, including unstacking of grana regions under heat stress in light. It was accompanied by the phosphorylation of thylakoid proteins such as D1 and D2 proteins and the light harvesting complex II proteins Lhcb1 and Lhcb2. These results suggest that heat stress at 40 °C in light induces state 1 to state 2 transition for the preferential excitation of photosystem I (PSI) by phosphorylating thylakoid proteins more strongly. Structural changes of thylakoid membrane also assist the remodeling of photosystems and regulation of energy distribution by transition toward state 2 probably contributes to plastoquione oxidation, thus, light-driven electrons flowing through PSI play a protective role against PSII damage under heat stress.

An endopeptidase was purified and characterized from green leaves of cucumber ( Cucumis sativus L. suyo). The purified enzyme, a basic amino acid-specific endopeptidase with a pI of 5.0, was a monomeric protein of 80 kDa whose pH optimum was 9.5. Inhibitor analysis suggested that it was a serine endopeptidase and contained sulfhydryl groups essential for catalytic activity. Analysis of internal amino acid sequences of the endopeptidase showed no significant similarity to other proteins. Its activity was inhibited by l -Arg and guanidino compounds having high hydrophobicity, as well as divalent cations such as Mg 2+ and Ca 2+ . The K i values of l -Arg and Mg 2+ , which are also likely in vivo inhibitors, were 3.5 and 10 mM, respectively. Inhibition by l -Arg and Mg 2+ was additive, and more than 70% of the activity was reversibly inhibited under their physiologically significant concentrations. These results suggest that the enzyme is possibly regulated by l -Arg and/or guanidino compounds, and by divalent cations in vivo.

In this study, a highly active foliar aminopeptidase preferentially releasing N-terminal alanine from artificial substrates was purified and characterized from cucumber (Cucumis sativus L. suyo). The enzyme had a molecular mass of 200 kDa consisting of two subunits of 95 kDa. It was a metalloprotease the pH optimum of which was 8 to 9. It cleaved Ala-, Gly-, Met-, Ser-, Leu-, Lys-, and Arg artificial substrates. An internal amino acid sequence was similar to those of aminopeptidase N (clan MA, family M1) of microorganisms, and was very similar to that of a putative aminopeptidase N of Arabidopsis thaliana. From these results, the highly active aminopeptidase in cucumber leaves was identified to be a plant aminopepitdase N.

This study investigated whether three biodegradable materials, poly–N–acetyl–D–glucosamine (chitin), poly-e-caprolactone (p-CL), polylactic acid (PLA), and chitin/p-CL composite could be used as scaffold implants in the reconstruction of extra-articular ligaments or tendons. Braided artificial tendons made from these materials were soaked in phosphate-buffered saline or implanted in the subcutaneous tissue of 31 Japanese white rabbits and then subjected to load at failure testing. There was no significant loss of load at failure when chitin tendons were soaked in saline for 26 weeks, but a rapid decrease occurred in vivo. The other two types of tendons showed significant loss of strength after 26 weeks both in vitro and in vivo. Another 111 rabbits were used to assess Achilles tendon reconstruction with the braided tendon implants. The tendon regeneration process was assessed macroscopically, histologically, immunohisto-chemically, and mechanically (maximum load at failure). Chitin showed more rapid degradation than the other materials on histological examination. There was good formation of fibrous tissue composed of type I and type III collagen around the chitin, PLA, and chitin/p-CL fibers in comparison with p-CL fibers, but chitin tendons showed more rapid loss of strength after implantation. Both PLA and the chitin/p-CL composite tendons had good initial strength and showed increased ingrowth of fibrous tissue, suggesting that these materials are promising as artificial tendons.

We investigated the time taken for a tear in the vascular portion of the meniscus to heal and whether free synovial grafting or the presence of basic fibroblast growth factor (bFGF) had a favorable effect on the reparatory process. Adult mongrel dogs were used, and a longitudinal tear, of about 5 mm, was made at the portion one-third of the periphery of the mid-portion of the medial meniscus. The animals were divided into three groups: free synovial graft group, in which a free synovial graft was performed; FGF group, in which animals were locally injected with bFGF at the peripheral synovium of the tear; and control group, in which animals the longitudinal tear was made, but no other procedures were performed. Animals were killed after various periods, and microangiograms and histological observations were made. In all groups, it took more than 16 weeks for the tear to heal. There was no significant histological difference between the free synovial graft group and the control group, but in the FGF group, chondrocyte-like round cells appeared earlier than in the other two groups, suggesting a favorable effect of bFGF on the reparatory process.

The degradation of storage protein in germinating cucumber seeds was shown to proceed via two distinct steps. First, several proteases with acidic isoelectric points (pIs) were involved in solubilization and partial degradation of 11S globulin. Treatment of seedlings with cycloheximide inhibited this step and the expression of these proteases. Thus, the first step appeared to be governed by these proteases, which were synthesized de novo after imbibition. The first step was observed in dark-grown cotyledons, but the complete degradation of 11S globulin did not occur in the absence of illumination. An additional protease, with a pI of 4.5, was induced by illumination, and it was involved in the further cleavage of the partially degraded products of 11S globulin. Thus, the complete degradation of the storage protein proceeded via a two-step process in illuminated germinating seedlings. Light is needed to induce the second step in the degradation of 11S globulin that supplies the nitrogen required for development of the photosynthetic apparatus in the greening cotyledon.

Adhesion molecules of the integrin family, including very late activation antigens (VLA), have been implicated in various cellular functions. In this study, we investigated the contribution of integrin-mediated interaction with ECM proteins to the cytokine gene expression in human chrondrocytes. Human articular chronrdocytes expressed VLA-1, -2, -3 and -5 on the cell surface, and could adhere to various ECM proteins, especially to fibronectin (FN). Futhermore, the production of GM-CSF and IL-6 was potently induced by culturing chondrocytes on immobilized FN. This stimulative effect of FN was completely inhibited by an anti-integrin α5 chain mAb, as well as by anti-integrin β1 chain mAbs. These results indicate an important role of the VLA-5-mediated interaction with FN in regulating inflammatory cytokine production by human articular chondrocytes.

Oxygenic phototrophs convert photon energy into chemical energy through a series of lightinduced electron transfer reactions initiated with charge separation of chlorophyll (Chl) special pairs located in the central part of photosystem I and II (PSI and PSII) (Fig. 1). The reducing power is transferred from PSII to PSI through cytochrome b6f, and finally utilized for reduction of NADP+ to assimilate CO2. The oxidized equivalents accumulated on the PSII donor side are neutralized by substrate water molecules to release protons for driving ATP synthase and O2 molecules as a by-product. This water oxidation takes place in the oxygen-evolving complex (OEC) of PSII [McEvoy & Brudvig, 2006, Renger & Renger, 2008]. The OEC assembly is largely similar between cyanobacteria and higher plants, except for a critical difference in the composition of extrinsic proteins [Roose et al., 2007]. In cyanobacteria, PsbO, PsbV, and PsbU residing on the lumenal side of PSII play significant roles in the regulation and stabilization of the water oxidation machinery. Higher plants possess major nuclear gene-encoded extrinsic proteins named PsbO, PsbP, and PsbQ. PsbO is a common extrinsic protein highly conserved among the oxygenic phototrophs. PsbP and PsbQ are indigenous to plant PSII and have been proposed as the functional equivalents of PsbV and PsbU in bacterial PSII, having replaced them during the course of evolution from ancestral cyanobacteria to higher plants. These proteins play significant roles in the regulation and stabilization of the photosynthetic water oxidation [Roose et al., 2007, Seidler, 1996, Williamson, 2008] although the details of their function(s) are still a matter of debate.

Acylamino acid-releasing enzyme/oxidized protein hydrolase (AARE/OPH) has been biochemically demonstrated to be a bifunctional protease that has exopeptidase activity against Nα-acylated peptides and endopeptidase activity against oxidized and glycated proteins; however, its physiological role remains unknown. In this study, to determine its physiological significance, we produced AARE/OPH-overexpressing and -suppressed plants and assessed the biological impacts of AARE/OPH. The subcellular localization of Arabidopsis AARE/OPH was found to be cytoplasmic and nuclear by transient expression analysis of tdTomato-fused Arabidopsis AARE/OPH. Overexpression of AARE/OPH exhibited no apparent effect on the level of oxidized proteins because wild types probably have inherently high AARE/OPH activity. Through RNAi gene suppressing, we successfully produced AARE/OPH-suppressed Arabidopsis plants (aare) that exhibited almost no AARE activity. In the aare plant, electrolyte leakage by methyl viologen treatment was enhanced compared to that of non-transformant plants, suggesting that the plasma membranes of aare easily suffered oxidative damage, probably as a result of deterioration of the cytoplasmic antioxidative system. Correspondingly, proteomic analysis revealed that the aare plant accumulated a number of oxidized proteins including cytoplasmic antioxidant enzymes. On the basis of these results, we concluded that AARE/OPH plays a homeostatic role in sustaining the cytoplasmic antioxidative system.

Plants are often exposed to temperatures of around 40^o^C. These temperatures can cause serious damage to photosystems, yet plants can survive with minimum damage. Here, we show that plants switch photosystem to protect photosystem II (PSII) at 40^o^C. Using wheat and Arabidopsis seedlings, we investigated the mechanisms of heat-derived damage in the dark and avoidance of damage in the light. Heat treatment at 40^o^C in the dark caused serious damage to PSII: the maximum quantum yield of PSII (Fv/Fm) and oxygen evolution rapidly decreased. The damage was due to the degradation of the D1 protein (shown by immuno-chemical analysis) and the disturbance of energy transfer in PSII core chlorophyll-binding proteins CP43 and CP47 (shown by time-resolved fluorescence measurement). The damage to PSII might be due to enhanced introduction of electrons from the reducing power of the stroma into thylakoid membranes, causing subsequent electron backflow to PSII. Plants treated at 40^o^C in the light avoided PSII damage and showed preferential excitation of photosystem I (PSI), phosphorylation and migration of light-harvesting complex II (LHCII), which indicate state transition of the photosystem to enhance thermal dispersion and light-driven cyclic electron flow around PSI. These results suggest that heat damage to PSII is probably due to a backflow of reducing power from the stroma to PSII, and that light causes a state transition of photosystem, driving cyclic electron flow and thus protecting PSII from damage.

Six hundred surgical cases of lumbar intervertebral disc herniation were evaluated histologically for the presence of blood vessels. These patients ranged in age from 12 to 77 years. Blood vessels were observed in 57 of 101 cases of complete prolapse type of herniated disc (56.4%), 12 of 32 cases of incomplete prolapse type of herniated disc (37.5%), and 53 of 467 cases of protrusion type of intervertebral disc herniation (11.3%). The presence of blood vessels in intervertebral discs was also investigated in postmortem specimens. Blood vessels were observed in 293 of 616 intervertebral discs (T10-L5), in individuals older than 40 years of age from 100 postmortem spines. The specimen age range was 16-89 years. Most of the blood vessels seen in the extruded tissue, exposed to the epidural space in cases of complete and incomplete prolapse type of herniation, are thought to have been newly formed after herniation occurred. As invasion of the intervertebral disc by blood vessels was found to occur with the advance of age, it is possible that such blood vessels become extruded with the intervertebral disc tissue. The intervertebral disc may herniate posteriorly in three basic patterns. The first pattern is "protrusion type of herniated disc." In protrusion hernia type there is abnormal posterior bulging of the anulus fibrosus. The disc pathology is predominantly nucleus pulposus, and the peripheral layer of the anulus fibrosus remains attached to the vertebral body bony rim, however.(ABSTRACT TRUNCATED AT 250 WORDS)

種々のリウマチ性疾患患者を対象にして,血漿中の補体分解産物C 4 d, iC 3 bおよびBb濃度をモノクローナル抗体を用いたEIAキットを用いて測定し,血清補体価,補体成分および免疫複合体量との関連を検討した.一般に低補体血症を示す全身性エリテマトーデスだけでなく,高補体血症を示す慢性関節リウマチや大動脈炎症候群, Behget病などの炎症性結合組織疾患でも血漿中のC 4 d, iC 3 bおよびBbが増加しており,とくにBbの増加するものが多くみられた.全身性エリテマトーデス,混合性結合組織病,多発性筋炎,強皮症および慢性関節リウマチでは補体分解産物の間に相関を認めなかったが,大動脈炎症候群とBehcet病においてC 4 dとiC 3 bとの間に正の相関を認めた.全身性エリテマトーデスにおいてC 4 dが免疫複合体量と正の,血清補体価と負の相関を示したが, iC 3 bとBbは免疫複合体量や血清補体価との相関を示さなかった.以上,種々のリウマチ性疾患において,免疫複合体による補体古典的経路の活性化だけでなく,補体第2経路の活性化をも生じていることが示唆された.今後,各々の症例で補体分解産物を測定することは,補体活性化の関与する病態の解明に有用であると思われる.

Dechlorodauricumine (5) and dechloroacutumine (6) were converted to miharumine (7) and dechloroacutumidine (8), respectively, by a cell-free preparation from cultured roots of Menispermum dauricum in the presence of FAD. The structures of 7 and 8 were elucidated on the basis of spectroscopic analyses and chemical conversion.

To elucidate the mechanism of vascular thrombosis in patients with systemic lupus erythematosus and the lupus anticoagulant changes in factors associated with haemostasis were investigated. The lupus anticoagulant was associated with an increased incidence of thrombosis, particularly cerebral thrombosis. Concentrations of fibrinopeptide A and fibrinopeptide B beta 15-42 were significantly raised in the plasma of patients with systemic lupus erythematosus and the anticoagulant compared with concentrations in patients without the lupus anticoagulant. The tendency towards formation of thrombosis was not found in all lupus patients with the anticoagulant, however. Concentrations of thromboxane B2 were remarkably raised in the plasma of the two patients with the lupus anticoagulant who had recently had thrombosis. Concentrations of 6-keto-prostaglandin F1 alpha, protein C, antithrombin III, and plasminogen were similar in both groups. No significant decrease in serum stimulatory activity on prostacyclin production by cultured aortic endothelial cells was noted in lupus patients with the anticoagulant, but inhibition was present in the two patients with recent thrombosis. These results indicate that although patients with the lupus anticoagulant are not always in a hypercoagulable state, haemostatic abnormalities found in some patients with the anticoagulant may be predictive of thrombotic events.

To determine whether there is an intrinsic defect in T cells from patients with systemic lupus erythematosus (SLE), we studied signal transduction systems, assaying the total protein kinase C (PKC) levels and the phorbol myristate acetate (PMA)-induced activation of PKC in PHA-treated T cells. T cells from SLE patients showed a decrease in proliferation in response to PMA, but not to PHA, thereby suggesting the existence of an intrinsic abnormality in the PKC-mediated activation pathway. Total PKC activity in the T cells from SLE patients was significantly decreased. Although stimulation with PMA induced a translocation of PKC from the cytosol to the particulate fraction, translocated PKC activity after 2 nM PMA treatment was decreased in the SLE T cells. Furthermore, PMA-induced phosphorylation of 80-kDa substrates was also decreased in SLE T cells. These results suggest that there is a reduced PKC activity and an impaired PKC activation in response to PMA in the SLE T cells, a finding which may explain, if partially, the defect in T cell activation in patients with SLE.

Mixed connective tissue disease (MCTD) is characterized as a benign rheumatic disease with a favorable response to therapy. When pulmonary hypertension is a complication, however, it is often reported to be fatal. A 32-year-old female patient with MCTD who had developed rapidly progressive pulmonary hypertension and disseminated intravascular coagulopathy was admitted to our hospital and was successfully treated with corticosteroids and anticoagulants. The failure of microcirculation due to coagulopathy is considered to be one of the possible entities of pulmonary hypertension.

We determined the incidence of herpes zoster (HZ) in 119 patients with systemic lupus erythematosus (SLE). HZ occurred in 56 patients (47%), and 9 patients had had HZ even before SLE developed. After diagnosis of SLE, an incidence of zoster was high, 5.45 cases per 100 person-years. It was found that the susceptibility to HZ was not related to the presence of renal disorder or maximum dose of corticosteroids. The patients with SLE who had had HZ showed significantly higher antibody titers than those without a history of HZ and normal subjects as assayed by both complement fixation technique and neutralization test. On the other had, only 17 of 55 patients (31%) with SLE showed positive skin reactions to varicella zoster virus (VZV) antigen, whereas all 15 normal subjects had positive reactions. In the patients who were receiving less than 10 mg/day of prednisolone, 11 of 17 (65%) had positive skin reactions to VZV antigen, whereas only 4 of 31 (13%) patients who were receiving 10 mg/day or more prednisolone showed positive reactions. It was of interest that in 7 patients with SLE who had not received corticosteroids, only 2 (29%) patients showed positive skin reactions to VZV antigen. These results suggest that high incidence of HZ in patients with SLE is probably due to an impaired cellular immunity because of both underlying disease and corticosteroid treatment.