Simultaneous analysis of amino acids and carboxylic acids by capillary electrophoresis-mass spectrometry using an acidic electrolyte and uncoated fused-silica capillary.

A simple, low-cost capillary electrophoresis-mass spectrometry (CE-MS) method is demonstrated for the simultaneous analysis of amino acids and small carboxylic acids (glycerate, lactate, fumarate, succinate, malate, tartrate, citrate, iso-citrate, cis-aconitate, and shikimate). All CE-MS experiments were performed using a single uncoated fused-silica capillary and with a single separation electrolyte, formic acid. For CE polarity, the CE inlet was set as the anode, and the MS side was set as the cathode. By using high-speed sheath gas flow, the apparent mobilities of all compounds were sped up; thus, the migration times of the carboxylic acids were reduced. In positive ion mode ESI-MS detection, small carboxylic acids were detected faintly as m/z = [M + 18](+) or [M + 23](+), after protonated molecule detection (m/z = [M + 1](+)) of the amino acids. In negative ion mode, all of these small carboxylic acids were detected clearly as deprotonated molecules (m/z = [M - 1](-)), after detection of the amino acids. By changing the polarity of the MS during CE separation, both amino acids and small carboxylic acids were detectable in a single electrophoresis analysis run. With this method, the diurnal metabolic changes of pineapple leaves were observed as reflecting Crassulacean acid metabolism.

Improvement of driver active interventions during automated driving by displaying trajectory pointers-A driving simulator study.

Objective: The handover of vehicle control from automated to manual operation is a critical aspect of interaction between drivers and automated driving systems (ADS). In some cases, it is possible that the ADS may fail to detect an object. In this event, the driver must be aware of the situation and resume control of the vehicle without assistance from the system. Consequently, the driver must fulfill the following 2 main roles while driving: (1) monitor the vehicle trajectory and surrounding traffic environment and (2) actively take over vehicle control if the driver identifies a potential issue along the trajectory. An effective human-machine interface (HMI) is required that enables the driver to fulfill these roles. This article proposes an HMI that constantly indicates the future position of the vehicle. Methods: This research used the Toyota Dynamic Driving Simulator to evaluate the effect of the proposed HMI and compares the proposed HMI with an HMI that notifies the driver when the vehicle trajectory changes. A total of 48 test subjects were divided into 2 groups of 24: One group used the HMI that constantly indicated the future position of the vehicle and the other group used the HMI that provided information when the vehicle trajectory changed. The following instructions were given to the test subjects: (1) to not hold the steering wheel and to allow the vehicle to drive itself, (2) to constantly monitor the surrounding traffic environment because the functions of the ADS are limited, and (3) to take over driving if necessary. The driving simulator experiments were composed of an initial 10-min acclimatization period and a 10-min evaluation period. Approximately 10 min after the start of the evaluation period, a scenario occurred in which the ADS failed to detect an object on the vehicle trajectory, potentially resulting in a collision if the driver did not actively take over control and manually avoid the object. Results: The collision avoidance rate of the HMI that constantly indicated the future position of the vehicle was higher than that of the HMI that notified the driver of trajectory changes, χ2 = 6.38, P < .05. The steering wheel hands-on and steering override timings were also faster with the proposed HMI (t test; P < .05). Conclusions: This research confirmed that constantly indicating the position of the vehicle several seconds in the future facilitates active driver intervention when an ADS is in operation.

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice.

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.

Leaf contents differ depending on the position in a rice leaf sheath during sink-source transition.

Carbohydrate contents varied with position in a leaf sheath, and differed between the flag leaf sheath and the second leaf sheath below the flag leaf (-2 leaf sheath) in rice (Oryza sativa L.). In the -2 leaf sheath before heading, light microscopy revealed differences in the distribution of starch granules depending on position. Leaf sheaths were divided into several parts, and the contents of carbohydrates (starch, sucrose, and hexoses) were measured in each part. Before heading, the content of accumulated starch increased linearly from the top to the bottom in -2 leaf sheaths (r2=0.99, P<0.001), as did the contents of accumulated sucrose and hexoses in flag leaf sheaths (r2=0.94, P<0.01). In flag leaf sheaths, the relative content of sucrose synthase (SuS), which plays a central role in the degradation of sucrose into hexoses, increased from the top to the bottom, consistent with hexose contents. After heading, the accumulated carbohydrates were dramatically decreased. In -2 leaf sheaths, the activity of alpha-amylase (EC 3.2.1.1), the rate-limiting step in starch degradation, was consistent with the degree of starch degradation, but in flag leaf sheaths with little starch before heading. These results show that carbohydrate contents differ, depending on the position in a leaf sheath. In addition, there were big differences in leaf contents between flag leaf sheaths and -2 leaf sheaths.

Impacts of acute ozone stress on superoxide dismutase (SOD) expression and reactive oxygen species (ROS) formation in rice leaves.

Tropospheric ozone is an air pollutant harmful to plants and animals. Its rapid increase at the ground surface has raised serious concern over damage to the quality and yield of agricultural crops. Reactive oxygen species (ROS) are formed in plant cells when that are exposed to a high concentration of ozone, and the ROS are thought to alter gene expression and result in cellular death. Clarification of the ROS formation will provide us with a better understanding of the physiological responses to and signaling pathways of ozone stress in plants. In this study, we investigated the mechanisms of the ROS formation in rice (Oryza sativa L.), the premier crop in Asia. To determine ROS distribution in rice leaves under acute ozone stress, we analyzed superoxide dismutase (SOD) expression, lipid peroxidation, NADPH oxidase activity, and in vivo H2O2 formation. Interestingly, chloroplastic, peroxisomal and mitochondrial SODs down-regulated their expression levels under ozone stress, whereas cytosolic SODs maintained their expression level. Higher lipid peroxidation occurred after the end of ozone exposure, which suggests lipid peroxidation may not be due to ozone directly, but rather to metabolic changes caused by the ozone exposure. Activity of NADPH oxidase did not show significant change. The in vivo analysis indicated accelerated formation of H2O2 about 24 h after the onset of exposure, which suggests that cellular death occurred around this time.

Expression patterns of genes encoding carbohydrate-metabolizing enzymes and their relationship to grain filling in rice (Oryza sativa L.): comparison of caryopses located at different positions in a panicle.

In rice, caryopses located at the base of the panicle have a lower growth rate than those at the tip of the panicle. The former and latter types of caryopses are called inferior and superior caryopses, respectively. Taking the different growth rate into consideration, sugar status and the expression of genes encoding carbohydrate-metabolizing enzymes in inferior caryopses were compared with those in superior caryopses. During the first 5 d after flowering, superior caryopses elongated rapidly, but inferior caryopses did not. At this phase, inferior caryopses had a low ratio of hexose to sucrose, high activity of acid invertase and the absence of the expression of the genes encoding the above enzymes except for two isoforms of cell wall invertase, OsCIN4 and INV1, in comparison with superior caryopses. At the start of caryopsis elongation in both superior and inferior caryopses, the hexose/sucrose ratio increased accompanied by gene expression of vacuolar invertase (INV3), sucrose synthase (RSus1) and ADP-glucose pyrophosphorylase (AGP-L2: D50317). Furthermore, the genes related to endospermal starch accumulation were expressed highly with the decrease in the hexose/sucrose ratio after its peak. Based on the comparison of superior and inferior caryopses, the possible mechanism of grain filling in rice is discussed.

Seasonal changes in canopy photosynthesis and respiration, and partitioning of photosynthate, in rice (Oryza sativa L.) grown under free-air CO2 enrichment.

An increase in atmospheric CO(2) concentration ( [CO(2)]) is generally expected to enhance photosynthesis and biomass. Rice plants (Oryza sativa L.) were grown in ambient CO(2) (AMB) or free-air CO(2)-enrichment (FACE), in which the target [CO(2)] was 200 micromol mol(-1) above AMB. (13)CO(2) was fed to the plants at different stages so we could examine the partitioning of photosynthates. Furthermore, canopy photosynthesis and respiration were measured at those stages. The ratio of (13)C content in the whole plant to the amount of fixed (13)C under FACE was similar to that under AMB at the vegetative stage. However, the ratio under FACE was greater than the ratio under AMB at the grain-filling stage. At the vegetative stage, plants grown under FACE had a larger biomass than those grown under AMB owing to enhancement of canopy photosynthesis by the increased [CO(2)]. On the other hand, at the grain-filling stage, CO(2) enrichment promoted the partitioning of photosynthate to ears, and plants grown under FACE had a greater weight of ears. However, enhancement of ear weight by CO(2) enrichment was not as great as that of biomass at the vegetative stage. Plants grown under FACE did not necessarily show higher canopy photosynthetic rates at the grain-filling stage. Therefore, we concluded that the ear weight did not increase as much as biomass at the vegetative stage owing to a loss of the advantage in CO(2) gain during the grain-filling period.

A rice heterochronic mutant, mori1, is defective in the juvenile-adult phase change.

We have identified five recessive allelic mutations, mori1-1 to mori1-5, which drastically modify the shoot architecture of rice. The most remarkable feature of mori1 plants is a rapid production of small leaves and short branches. The mori1 plants are about 5 cm in height even 7 months after sowing. No reproductive growth was attained in mori1 plants even if inductive short-day treatment was applied. Leaves of mori1 at any position were very small and the size and shape were comparable to those of the wild-type 2nd leaf. The stem of mori1 7 months after sowing did not differentiate node and internode and had randomly oriented vascular bundles, which were characteristic of the basal part of the wild-type stem where 2nd and 3rd leaves were inserted. These structural characteristics indicate that mori1 maintains the 2nd-leaf stage (juvenile phase) of the wild type. The short plastochron and high cell division activity in the shoot apical meristem further confirmed the juvenility of mori1, corresponding to the 2nd-leaf-differentiation stage in the wild-type embryo. Furthermore, the apparent photosynthetic rate in mori1 leaves was low as in the wild-type 2nd leaf. Thus, mori1 is a heterochronic mutation that suppresses the induction of adult phase and the termination of the juvenile phase. Therefore, MORI1 plays an important role in the juvenile-adult phase change. The importance of heterochronic mutations in modifying shoot architecture is discussed.

Uptake of atmospheric carbon dioxide into silk fiber by silkworms.

The relation between the uptake of atmospheric CO(2) and insect's production of silk fiber has not yet been reported. Here, we provide the first quantitative demonstrations that four species of silkworms (Bombyx mori, Samia cynthia ricini, Antheraea pernyi, and Antheraea yamamai) and a silk-producing spider (Nephila clavata) incorporate atmospheric CO(2) into their silk fibers. The abundance of (13)C incorporated from the environment was determined by mass spectrometry and (13)C NMR measurements. Atmospheric CO(2) was incorporated into the silk fibers in the carbonyl groups of alanine, aspartic acid, serine, and glycine and the C(gamma) of aspartic acid. We show a simple model for the uptake of atmospheric CO(2) by silkworms. These results will demonstrate that silkworm has incorporated atmospheric CO(2) into silk fiber via the TCA cycle; however, the magnitude of uptake into the silk fibers is smaller than that consumed by the photosynthesis in trees and coral reefs.