Akihiro Nose

Overexpression of C4 PEPC caused O2-insensitive photosynthesis in transgenic rice plants

We have introduced an intact maize gene for phosphoenolpyruvate carboxylase (PEPC) into C3 plants, rice (Oryza sativa cv. Kitaake). Most transgenic rice plants showed high-level expression of the maize gene. PEPC was two to three times more active in the leaves of some transgenic plants than in maize leaves. In transformants, the sensitivity of photosynthesis to O2 inhibition was reduced with the increased activity of the maize C4 PEPC. However, the alleviation of O2 inhibition was not due to an increase in the partial direct fixation of atmospheric CO2 via the enhanced maize PEPC, but rather due to the reduced stimulation of photosynthesis with a subatmospheric O2 level. Pi feeding to the leaves restored CO2 assimilation rate under the subatmospheric O2 condition, and consequently, the O2 inhibition in the transformants increased to a level comparable to that of the non-transformants. These results suggested that the O2-insensitive photosynthesis in the PEPC transformants was caused by a Pi limitation of photosynthesis. The activities of two key enzymes for sucrose synthesis, SPS and FBPase, and the sucrose and starch content were reduced in the leaves of the transformants. On the other hand, the dark respiration rate and the malate content in the leaves increased in the transformants. These results indicated that enhanced PEPC activity led to a decrease in the availability of Pi in chloroplast via a reduction of the activities of the key enzymes responsible for Pi recycling and it also caused an increased consumption of the substrate (triose-phosphate) in respiration, but not in sucrose biosynthesis.

Changes in the Contents of Metabolites and Enzyme Activities in Rice Plants Responding to Rhizoctonia solani Kuhn Infection: Activation of Glycolysis and Connection to Phenylpropanoid Pathway

Rhizoctonia solani Kuhn causes sheath blight disease in rice, and genetic resistance against it is the most desirable characteristic. Current improvement efforts are based on analysis of polygenic quantitative trait loci (QTLs), but interpretation is limited by the lack of information on the changes in metabolic pathways. Our previous studies linked activation of the glycolytic pathway to enhanced generation of lignin in the phenylpropanoid pathway. The current studies investigated the regulation of glycolysis by examining the time course of changes in enzymatic activities and metabolite contents. The results showed that the activities of all glycolytic enzymes as well as fructose-6-phosphate (F-6-P), fructose-1,6-bisphosphate (F-1,6-P(2)), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP) and pyruvate contents increased. These results combined with our previous findings that the expression of phosphoglucomutase (PGM), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and pyruvate kinase (PK) increased after infection suggested that the additional establishment of glycolysis in the cytosol compartment occurred after infection. Further evidence for this was our recent findings that the increase in expression of the 6-phosphofructokinase (PFK) plastid isozyme Os06g05860 was accompanied by an increase in expression of three cytosolic PFK isozymes, i.e. Os01g09570, Os01g53680 and Os04g39420, as well as pyrophosphate-dependent phosphofrucokinase (PFP) isozymes Os08g25720 (α-subunit) and Os06g13810 (β-subunit) in infected rice plants of the resistant line. The results also showed that the reactions catalysed by PFK/PFP, aldolase, GAPDH + phosphoglycerate kinase (PGK) and PK in leaf sheaths of R. solani-infected rice plants were non-equilibrium reactions in vivo. This study showed that PGM, phosphoglucose isomerase (PGI), TPI and phosphoglycerate mutase (PGmu) + enolase could be regulated through coarse control whereas, PFK/PFP, aldolase, GAPDH + PGK and PK could be regulated through coarse and fine controls simultaneously.

Infection of Rice Plants with the Sheath Blight Fungus Causes an Activation of Pentose Phosphate and Glycolytic Pathways

The response of key regulatory enzymes of the pentose phosphate and glycolytic pathways in disease development was assessed in genetically-related rice plants resistant and susceptible to the sheath blight fungus, Rhizoctonia solani. The plants were grown and maintained under greenhouse conditions and inoculated at 50% flowering. Uninoculated healthy plants served as controls. The activities of pentose phosphate pathway enzymes (glucose-6 phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) increased more than two-fold in both the resistant and susceptible plants. Activities of ATP- and pyrophosphate-dependent phosphofructokinase and phosphoenolpyruvate phosphatase increased in infected plants while activity of phosphoenolpyruvate carboxylase in infected plants was lower than in the healthy plants. Furthermore, for enzymes with increased activity, the levels were higher in the resistant line than in the susceptible line. The enhancement of the enzyme activities correlated well with the post infection period. These data suggest that altered carbohydrate metabolism in sheath blight infections may play an important role in modulating the rice plants response to infection. The isolation of an infection-induced gene encoding a basic enzyme of pentose phosphate and glycolytic pathways could be used to develop plants with more resistance towards sheath blight disease.

Potential of the Common Ice Plant, Mesembryanthemum crystallinum as a New High-Functional Food as Evaluated by Polyol Accumulation

Abstract We measured the concentration of polyols (pinitol, ononitol, and myo-inositol), which are known to have health-promoting and/or disease-preventing functions, in the common ice plant (Mesembryanthemum crystallinum L.) cultured under salt- and drought-stressed treatments. In NaCl-treated plant the concentration of pinitol/ononitol increased with increasing NaCl concentration in culture solution. The maximal concentration was 3.6 mg g-1 FW, which was foundin the shoot top, followed by small side shoots (2.1 mg g-1 FW) of mature plants grown with 400 mM NaCl for 35 ds. The drought stress also accelerated the accumulation of pinitol/ ononitol. The maximal concentration was 1.2 mg g-1 FW, which was found in the shoot top of plants under the stress for 25 ds. The myo-inositol increased in salt-stressed plants at 3 ds after the start of the treatment and then decreased with the lapse of time during stress. The concentration of polyols in the ice plant was comparable to that in the other species reported to accumulate polyols at high levels. Radical scavenging activity evaluated by DPPH assay was increased two-fold by 400 mM NaCl treatment, which was twice as high as that in the leaves of lettuce (Lactuca sativa L.). These results indicated the high potential of the ice plant as a polyol-rich high-functional food.

Effects of Chilling Temperature on the Activity of Enzymes of Sucrose Synthesis and the Accumulation of Saccharides in Leaves of Three Sugarcane Cultivars Differing in Cold Sensitivity

The effects of short-term exposure to chilling temperature (10 °C) on sucrose synthesis in leaves of the cold-tolerant sugarcane cultivars Saccharum sinense R. cv. Yomitanzan and Saccharum sp. cv. NiF4, and the cold-sensitive cultivar S. officinarum L. cv. Badila were studied. Plants were grown at day/night temperatures of 30/25 °C, and then shifted to a constant day/night temperature of 10 °C. After 52-h exposure to the chilling temperature, sucrose content in the leaves of NiF4 and Yomitanzan showed a 2.5- to 3.5-fold increase relative to that of the control plants that had been left on day/night temperatures of 30/25 °C. No such increase was observed in Badila leaves. Similarly, starch content in the leaves of NiF4 and Yomitanzan was maintained high, but starch was depleted in Badila leaves after the 52-h exposure. During the chilling temperature, sucrose phosphate synthase (SPS; E.C.2.4.1.14) activity was relatively stable in the leaves of NiF4 and Yomitanzan, whereas in Badila leaves SPS activity significantly decreased. There was no significant change in cytosolic fructose-1,6-bisphosphatase activity for the three cultivars at the chilling temperature. This supports the hypothesis that: (1) on exposure to chilling temperature, sucrose content in sugarcane leaves is determined by the photosynthetic rate in the leaves, and is not related to SPS activity; (2) SPS activity in sugarcane leaves at chilling temperature is to be determined by sugar concentration in the leaves.

Effect of urea-type cytokinins on the adventitious shoots regeneration from cotyledonary node explant in the common ice plant, Mesembryanthemum crystallinum

Abstract Mesembryanthemum Crystallinum (Common Ice Plant) Was Used As A Model Plant To Study The Regulatory Properties of Crassulacean Acid Metabolism (Cam) and Tolerance To Abiotic Stresses. Although Transformation Is A Useful Genetic Approach, It Has Not Been Established in This Species Due To Recalcitrancy For Regeneration. To Establish An Efficient Procedure For Regeneration of M. Crystallinum, We Examined The Effects of Urea-Type Cytokinins, Thidiazuron (Tdz) and Forchlorofenuron (Cppu) On The Adventitious Shoot induction. Adventitious Shoots Were Generated Only From Explants Obtained From The Cotyledonary Node, Not From Explants Obtained From The Cotyledon, Hypocotyl and Roots. Urea-Type Cytokinins, Tdz and Cppu Were More Effective For The induction and The Morphogenesis of Adventitious Shoots Than Adenine-Type Cytokinin, 6-Benzyladenopurine (Ba). We Have Found That The 2.5 Mg L-1 Tdz induced The Largest Number of Multiple Shoots and The Highest Frequency of Adventitious Shoot induction From Single Explant. in Addition, Fewer Hyperhydric Shoots Were Produced On The Medium Containing Tdz Than in That Containing Ba and Cppu in The Presence of 1.0 Mg L-1 Naa. The Regenerated Shoots Rooted On The Ms Medium Within One Month, and The Rooting Was Promoted By Replacing The Agar Medium With Vermiculite Or Florialite. The Fertile Plant With Normal Morphological Properties Was Harvested For Four Months After Sowing. Using The Improved Regeneration Procedure With Tdz, We Successfully introduced A Kanamycin-Resistant Gene (Nptii-Hph) into The Cotyledonary Node Mediated By Agrobacterium Tumefaciens. These Results indicated That This Regeneration Procedure Using Cotyledonary Node Explants and Tdz Could Be Useful For The Genetic Engineering of M. Crystallinum.

Species variation in the intracellular localization of pyruvate, Pi dikinase in leaves of crassulacean-acid-metabolism plants: an immunogold electron-microscope study.

Abstract. In malic enzyme-dependent crassulacean-acid-metabolism (ME-CAM) plants, malic acid is decarboxylated by NADP-ME and NAD-ME and generates pyruvate with CO2. Pyruvate is phosphorylated to phosphoenolpyruvate by pyruvate, Pi dikinase (PPDK) and is then conserved in gluconeogenesis. Although PPDK was considered to be located in chloroplasts (e.g., Mesembryanthemum crystallinum), it has recently been found to accumulate in both the chloroplasts and the cytosol in two Kalanchoë species. In this study, the intracellular localization of PPDK was investigated in 22 ME-CAM species in 13 genera of 5 families by immunogold labeling and electron microscopy. This revealed that the pattern of intracellular localization of PPDK varies among the ME-CAM plants and is divided into three types: Chlt, in which PPDK accumulates only in the chloroplasts; Cyt-Chlt, in which PPDK accumulates in both chloroplasts and cytosol; and Cyt, in which PPDK accumulates predominantly in the cytosol. Members of a particular genus tend to have a common PPDK-localization type. In the Cactaceae, all species from seven genera were classified as Cyt. The photosynthetic tissues of all ME-CAM species, including the Cyt type, had substantial PPDK activity, suggesting that PPDK in the cytosol is active and probably plays a functional role. In the Chlt species, NADP-ME activity was relatively greater than NAD-ME activity. In the Cyt-Chlt and Cyt species, however, either the activity of NAD-ME was higher than that of NADP-ME or they were approximately the same. The species variation in the intracellular localization of PPDK is discussed in relation to CAM function and to molecular and phylogenetic aspects.

Diurnal changes in photosynthesis in sugarcane leaves: II. Enzyme activities and metabolite levels relating to sucrose and starch metabolism.

Summary Diurnal changes in carbohydrate contents, assimilate export, enzyme activities and metabolite levels associated with sucrose and starch synthesis in field-grown sugarcane (Saccharum sp. cv. NiF4) leaves were investigated during a natural 24 h day-night cycle. Eighty percent of the carbon fixed in sugarcane leaves at midday was exported immediately. Of the total carbon fixed during the day, 82% was exported in the daytime and 17% was accumulated as leaf starch which was exported at night. The activities of sucrose phosphate synthase (SPS), cytosolic fructose-1, 6-bisphosphatase (FBPase), UDP-glucose pyrophosphorylase (UDPG-PPase) and ADP-glucose pyrophosphorylase (ADPG-PPase), and the levels of triose phosphates (triose-P) and fructose-1,6-bisphosphate (FBP) showed distinct diurnal fluctuations during the day-night cycle. The levels of hexose phosphates fluctuated at smaller magnitudes compared with those in triose-P and FBP. The diurnal change in triose-P level was highly related to the changes in carbon exchange rate and sucrose content, while the sucrose content was closely related to the changes in activities of SPS, cytosolic FBPase and UDPG-PPase in leaves of sugarcane. The present results suggest that the availability of triose-P is a key factor in determining the rate of sucrose synthesis in sugarcane leaves.

Gas Exchange Analysis for Estimating Net CO2 Fixation Capacity of Mangrove (Rhizophora stylosa) Forest in the Mouth of River Fukido, Ishigaki Island, Japan

Abstract Mangrove trees have been considered to possess a higher carbon fixation capacity than terrestrial trees although a reliable method to estimate their CO2 fixation capacity has not been established. In this study, net CO2 fixation in above-ground of Rhizophora stylosa was estimated as the difference between photosynthetic absorption and respiratory emission of CO2. In order to estimate these parameters, photosynthetic rates of single-leaves in response to light and temperature and the respiratory rates of leaves and branches in response to temperature were measured. Furthermore, we established a model of diurnal change in temperature. Monthly averages of the diurnal temperature change were used for correcting the CO2 absorption and emission. The effect of temperature modification on the estimation of net CO2 fixation was examined, and the net CO2 fixation capacity estimated with and without temperature modification was compared. Biomass accumulation estimated without temperature modification (i.e. corrected only for the light intensity) was 6.1 tons ha-1 yr-1, while that estimated with temperature modification (i.e. corrected for both light intensity and temperature) was 13.0 tons ha-1 yr-1. A doubling of the estimated values of net CO2 fixation as observed in this study was caused by the decrease in respiratory CO2 emission by half, which results from temperature modification. These findings suggest that temperature modification in gas exchange analysis could improve the accuracy of estimation of the net CO2 fixation capacity.

Effects of KCN, SHAM and Oxygen Concentrations on Respiratory Properties of Purified Mitochondria Isolated from Ananas comosus (Pineapple) and Kalanchoë daigremontiana

Abstract Effects of potassium cyanide (KCN), salicylhydroxamic acid (SHAM), and oxygen concentrations on mitochondrial respiration were investigated in purified mitochondria of a typical phosphoenolpyruvate carboxykinase (PCK) crassulacean acid metabolism (CAM) plant Ananas comosus (pineapple) and a typical malic enzyme (ME)-CAM plant Kalanchoë daigremontiana. Mitochondria of A. comosus oxidized succinate and NADH faster than that of K. daigremontiana. Succinate and NADH oxidations in mitochondria of both species were partially inhibited by KCN and SHAM, indicating that these oxidations were connected to cytochrome and alternative pathways in their electron transport chain (ETC). NADH oxidation was more sensitive to KCN than succinate oxidation, suggesting that the ETC from NADH oxidation was less connected to the alternative pathway than that from succinate in mitochondria of both species. Concurrent oxidation of succinate and NADH resulted in much higher rates of cytochrome and alternative respirations than each individual oxidation alone in both species. NADH oxidation in A. comosus mitochondria was more connected to the cytochrome pathway, so A. comosus could produce much more ATP than K. daigremontiana. This capacity might be one of the fitting mechanisms of A. comosus to produce a sufficient amount of ATP for cytosolic PCK in the daytime. In addition, the reduction of oxygen concentrations decreased not only the cytochrome respiration, but also the alternative respiration on succinate oxidation in mitochondria of both species, and the decrease was greater in K. daigremontiana than in A. comosus.