Different acyl-CoA:diacylglycerol acyltransferases vary widely in function, and a targeted amino acid substitution enhances oil accumulation.

Triacylglycerols (TAGs) are the major component of plant storage lipids such as oils. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and is mainly responsible for plant oil accumulation. We previously found that the activity of Vernonia DGAT1 was distinctively higher than that of Arabidopsis and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor bean were introduced into Arabidopsis. All Vernonia DGAT1-expressing lines showed a significantly higher oil content (49% mean increase compared with the wild-type) followed by soybean and castor bean. Most Arabidopsis DGAT1-overexpressing lines did not show a significant increase. In addition to these four DGAT1 genes, sunflower, Jatropha, and sesame DGAT1 genes were introduced into a TAG biosynthesis-defective yeast mutant. In the yeast expression culture, DGAT1s from Arabidopsis, castor bean, and soybean only slightly increased the TAG content; however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame increased TAG content >10-fold more than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions were created by site-directed mutagenesis and substantially increased the TAG content.

Acoustic streaming induced by MHz-frequency ultrasound extends the volume limit of cell suspension culture.

Large-scale cell suspension culture technology opens up opportunities for numerous medical and bioengineering applications. For these purposes, scale-up of the culture system is paramount. For initial small-scale culture, a simple static suspension culture (SSC) is generally employed. However, cell sedimentation due to the lack of agitation limits the culture volume feasible for SSC. Thus, when scaling up, cell suspensions must be manually transferred from the culture flask to another vessel suitable for agitation, which increases the risk of contamination and human error. Ideally, the number of culture transfer steps should be kept to a minimum. The present study describes the fabrication of an ultrasonic suspension culture system that stirs cell suspensions with the use of acoustic streaming generated by ultrasound irradiation at a MHz frequency. This system was applied to 100-mL suspension cultures of Chinese hamster ovary cells-a volume ten-fold larger than that generally used. The cell proliferation rate in this system was 1.88/day when applying an input voltage of 40 V to the ultrasonic transducer, while that of the SSC was 1.14/day. Hence, the proposed method can extend the volume limit of static cell suspension cultures, thereby reducing the number of cell culture transfer steps.

CO2 -responsive CCT protein interacts with 14-3-3 proteins and controls the expression of starch synthesis-related genes.

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 provides important findings regarding the molecular function of CRCT to control the expression of key starch synthesis-related genes.

Plutonium dioxide particle imaging using a high-resolution alpha imager for radiation protection.

The internal exposure of workers who inhale plutonium dioxide particles in nuclear facilities is a crucial matter for human protection from radiation. To determine the activity median aerodynamic diameter values at the working sites of nuclear facilities in real time, we developed a high-resolution alpha imager using a ZnS(Ag) scintillator sheet, an optical microscope, and an electron-multiplying charge-coupled device camera. Then, we designed and applied a setup to measure a plutonium dioxide particle and identify the locations of the individual alpha particles in real time. Employing a Gaussian fitting, we evaluated the average spatial resolution of the multiple alpha particles was evaluated to be 16.2 ± 2.2 µmFWHM with a zoom range of 5 ×. Also, the spatial resolution for the plutonium dioxide particle was 302.7 ± 4.6 µmFWHM due to the distance between the plutonium dioxide particle and the ZnS(Ag) scintillator. The influence of beta particles was negligible, and alpha particles were discernible in the alpha-beta particle contamination. The equivalent volume diameter of the plutonium dioxide particle was calculated from the measured count rate. These results indicate that the developed alpha imager is effective in the plutonium dioxide particle measurements at the working sites of nuclear facilities for internal exposure dose evaluation.

Suspension culture in a T-flask with acoustic flow induced by ultrasonic irradiation.

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.

Addition of luminescence process in Monte Carlo simulation to precisely estimate the light emitted from water during proton and carbon-ion irradiation.

Although luminescence of water lower in energy than the Cerenkov-light threshold during proton and carbon-ion irradiation has been found, the phenomenon has not yet been implemented for Monte Carlo simulations. The results provided by the simulations lead to misunderstandings of the physical phenomenon in optical imaging of water during proton and carbon-ion irradiation. To solve the problems, as well as to clarify the light production of the luminescence of water, we modified a Monte Carlo simulation code to include the light production from the luminescence of water and compared them with the experimental results of luminescence imaging of water. We used GEANT4 for the simulation of emitted light from water during proton and carbon-ion irradiation. We used the light production from the luminescence of water using the scintillation process in GEANT4 while those of Cerenkov light from the secondary electrons and prompt gamma photons in water were also included in the simulation. The modified simulation results showed similar depth profiles to those of the measured data for both proton and carbon-ion. When the light production of 0.1 photons/MeV was used for the luminescence of water in the simulation, the simulated depth profiles showed the best match to those of the measured results for both the proton and carbon-ion compared with those used for smaller and larger numbers of photons/MeV. We could successively obtain the simulated depth profiles that were basically the same as the experimental data by using GEANT4 when we assumed the light production by the luminescence of water. Our results confirmed that the inclusion of the luminescence of water in Monte Carlo simulation is indispensable to calculate the precise light distribution in water during irradiation of proton and carbon-ion.

Expression level of Rubisco activase negatively correlates with Rubisco content in transgenic rice.

The relationship between ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and Rubisco activase (Rca) levels was studied using transgenic rice overexpressing maize Rca (OX-mRca) and knockdown transgenic rice expressing antisense Rca (KD-Rca). The ratio of Rubisco to total soluble protein was lower in OX-mRca, whereas it was higher in KD-Rca than in WT, indicating that Rca expression was negatively correlated with Rubisco content. The expressions of other Calvin-Benson-Bassham cycle enzymes such as sedoheptulose-1,7-bisphosphatase and phosphoribulokinase analyzed by immunoblotting did not show such a negative correlation with Rca, suggesting that the effect of Rca on protein expression may be specific for Rubisco. Although Rubisco content was decreased in OX-mRca, the transcript levels of the Rubisco large subunit (OsRbcL) and the Rubisco small subunit mostly increased in OX-mRca as well as in KD-Rca. Additionally, polysome loading of OsRbcL was slightly higher in OX-mRca than it was in WT, suggesting that the OsRbcL translation activity was likely stimulated by overexpression of Rca. 35S-methionine labeling experiments demonstrated that there was no significant difference in the stability of newly synthesized Rubisco among genotypes. However, 35S-methionine-labeled Rubisco was marginally decreased in OX-mRca and increased in KD-Rca compared to the WT. These results suggest that Rca negatively affects the Rubisco content, possibly in the synthesis step.

Ethylene is not involved in adaptive responses to flooding in the Amazonian wild rice species Oryza grandiglumis.

The Amazonian wild rice Oryza grandiglumis has two contrasting adaptation mechanisms to flooding submergence: a quiescence response to complete submergence at the seedling stage and an escape response based on internodal elongation to partial submergence at the mature stage. We investigated possible factors that trigger these responses. In stem segments excised from mature O. grandiglumis plants, complete submergence only slightly promoted internodal elongation with increased ethylene levels in the internodes, while partial submergence substantially promoted internodal elongation without increased ethylene levels in the internodes. Incubation of non-submerged stem segments under a continuous flow of humidified ethylene-free air promoted internodal elongation to the same extent as that observed for partially submerged segments. Applied ethylene had little effect on the internodal elongation of non-submerged segments irrespective of humidity conditions. These results indicate that the enhanced internodal elongation of submerged O. grandiglumis plants is not triggered by ethylene accumulated during submergence but by the moist surroundings provided by submergence. The growth of shoots in O. grandiglumis seedlings was not promoted by ethylene or complete submergence, as is the case in O. sativa cultivars possessing the submergence-tolerant gene SUB1A. However, because the genome of O. grandiglumis lacks the SUB1A gene, the quiescence response of O. grandiglumis seedlings to complete submergence may be regulated by a mechanism distinct from that involved in the response of submergence-tolerant O. sativa cultivars.

Growth promotion and inhibition of the Amazonian wild rice species Oryza grandiglumis to survive flooding.

In Asian cultivated rice (Oryza sativa), distinct mechanisms to survive flooding are activated in two groups of varieties. Submergence-tolerant rice varieties possessing the SUBMERGENCE1A (SUB1A) gene display reduced growth during flash floods at the seedling stage and resume growth after the flood recedes, whereas deepwater rice varieties possessing the SNORKEL1 (SK1) and SNORKEL2 (SK2) genes display enhanced growth based on internodal elongation during prolonged submergence at the mature stage. In this study, we investigated the occurrence of these growth responses to submergence in the wild rice species Oryza grandiglumis, which is native to the Amazon floodplains. When subjected to gradual submergence, adult plants of O. grandiglumis accessions showed enhanced internodal elongation with rising water level and their growth response closely resembled that of deepwater varieties of O. sativa with high floating capacity. On the other hand, when subjected to complete submergence, seedlings of O. grandiglumis accessions displayed reduced shoot growth and resumed normal growth after desubmergence, similar to the response of submergence-tolerant varieties of O. sativa. Neither SUB1A nor the SK genes were detected in the O. grandiglumis accessions. These results indicate that the O. grandiglumis accessions are capable of adapting successfully to flooding by activating two contrasting mechanisms as the situation demands and that each mechanism of adaptation to flooding is not mediated by SUB1A or the SK genes.

Anoxia promotes gravitropic curvature in rice pulvini but inhibits it in wheat and oat pulvini.

Gravitropic curvature of pulvini of wheat and oat stem segments gradually declined with decreasing atmospheric O2 concentration and was almost completely blocked under anoxia, whereas that of rice stem segments was enhanced under hypoxia and anoxia. Anoxia substantially increased the ethanol content in pulvini of gravistimulated stem segments in rice, wheat and oat, but the ethanol content showed no marked difference between rice pulvini and wheat and oat pulvini. The concentrations of exogenous ethanol and acetaldehyde required to inhibit the gravitropic curvature of pulvini were significantly higher in rice segments than in wheat and oat segments. However, in all three species, the concentrations of ethanol and acetaldehyde required to completely inhibit curvature were several-fold higher than the endogenous levels that accumulated in pulvini gravistimulated in N2. The pulvini of rice segments gravistimulated in N2 did not contain much more ATP than those of wheat or oat segments gravistimulated in N2. When applied unilaterally to the pulvini of vertically oriented stem segments incubated in N2, indole-3-acetic acid induced bending in rice stem segments but not in wheat and oat stem segments. Transference of graviresponsive pulvini of rice, as well as those of wheat and oat, from aerobic conditions to anaerobic conditions led to cessation of gravitropic curvature within several minutes, but subsequently only gravitropic curvature of anoxic rice pulvini was completely recovered within 2 h. A large portion of this recovery was blocked by cordycepin, a transcription inhibitor. These results suggested that anoxia-induced expression of any gene or genes enables rice pulvini to respond to gravistimulation under anaerobic conditions, and that such a gene or genes might be unrelated to ethanol fermentation and ATP production in anaerobic conditions.

Involvement of cell wall-bound phenolic acids in decrease in cell wall susceptibility to expansins during the cessation of rapid growth in internodes of floating rice.

The cell walls in the elongating zone of submerged floating rice internodes show high susceptibility to expansins. When internode sections corresponding to such an elongation zone were incubated for 24h under osmotic stress conditions produced by treatment with 100mM polyethylene glycol 4000 (PEG), the cell wall susceptibility to expansins remained at its initial level, while the susceptibility of internode sections incubated under unstressed conditions decreased considerably during the same period. The contents of polysaccharides and phenolic acids as ferulic, diferulic and p-coumaric acids in the cell walls of internode sections increased substantially under unstressed conditions, but the increases were almost completely prevented by osmotic stress. Ferulic acid applied to internode sections under osmotic stress reduced the susceptibility of the cell walls to expansins and increased the levels of ferulic and diferulic acids in the cell walls, with little effect on the accumulation of polysaccharides. In contrast, applied p-coumaric acid increased the level of p-coumaric acid in the cell walls without a change in the levels of ferulic and diferulic acids but did not reduce the susceptibility to expansins. These results suggest that the deposition of ferulic and diferulic acids is a primary determinant in regulating the reduction of the susceptibility of cell walls to expansins in floating rice internodes.

Suppression and promotion of growth by ethylene in rice seedlings depends on ambient humidity.

We examined the effect of ethylene on the growth of rice seedlings (Oryza sativa L.) at various degrees of humidity. Ethylene significantly suppressed the growth of shoots when applied to seedlings grown under 30% relative humidity (RH), but promoted the growth of shoots when applied to seedlings grown under 100% RH. The application of gibberellic acid (GA(3)) promoted the elongation of shoots in seedlings grown under 30% and 100% RH. Ethylene inhibited the shoot elongation induced by GA(3) at 30% RH, but enhanced the elongation induced by GA(3) at 100% RH. These results indicate that ethylene can either promote or suppress the growth of rice shoots depending on ambient humidity, and that these actions of ethylene may be mediated through modulating the responsiveness of shoots to gibberellin.

Saturated humidity accelerates lateral root development in rice (Oryza sativa L.) seedlings by increasing phloem-based auxin transport.

Auxin transport plays a significant role modifying plant growth and development in response to environmental signals such as light and gravity. However, the effect of humidity on auxin transport is rarely documented. It is shown here that the transport of labelled indole-3-acetic acid (IAA) from the shoot to the root is accelerated in rice (Oryza sativa L. ssp. indica cv. IR8) seedlings grown under saturated humidity (SH-seedlings) compared with plants grown under normal humidity (NH-seedlings). The development of lateral roots in SH-seedlings was greatly enhanced compared with NH-seedlings. Removal of the shoot from SH-seedlings reduced the density of lateral roots, and the application of IAA to the cut stem restored the lateral root density, while the decapitation of NH-seedlings did not alter lateral root development. Phloem-based auxin transport appeared responsible for enhanced lateral root formation in SH-seedlings since (i) the rate of IAA transport from the shoot to the root tip was greater than 3.5 cm h-1 and (ii) naphthylphthalamic acid (NPA)-induced reduction of polar auxin transport in the shoot did not influence the number of lateral roots in SH-seedlings. It is proposed that high humidity conditions accelerate the phloem-based transport of IAA from the leaf to the root, resulting in an increase in the number of lateral roots.

Posttranscriptional regulation by the upstream open reading frame of the phosphoethanolamine N-methyltransferase gene.

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5' untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5' UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the beta-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream beta-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1-AtMEK1 pathway by wounding.

We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Delta166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Delta166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1-AtMEK1 pathway induced by wounding.

Similar regulation patterns of choline monooxygenase, phosphoethanolamine N-methyltransferase and S-adenosyl-L-methionine synthetase in leaves of the halophyte Atriplex nummularia L.

Glycinebetaine (betaine) highly accumulates as a compatible solute in certain plants and has been considered to play a role in the protection from salt stress. The betaine biosynthesis pathway of betaine-accumulating plants involves choline monooxygenase (CMO) as the key enzyme and phosphoethanolamine N-methyltransferase (PEAMT), which require S-adenosyl-L-methionine (SAM) as a methyl donor. SAM is synthesized by SAM synthetase (SAMS), and is needed not only for betaine synthesis but also for the synthesis of other compounds, especially lignin. We cloned CMO, PEAMT and SAMS isogenes from a halophyte Atriplex nummularia L. (Chenopodiaceous). The transcript and protein levels of CMO were much higher in leaves and stems than in roots, suggesting that betaine is synthesized mainly in the shoot. The regulation patterns of transcripts for SAMS and PEAMT highly resembled that of CMO in the leaves during and after relief from salt stress, and on a diurnal rhythm. In the leaves, the betaine content was increased but the lignin content was not changed by salt stress. These results suggest that the transcript levels of SAMS are co-regulated with those of PEAMT and CMO to supply SAM for betaine synthesis in the leaves.

Enhancement of transpiration by ethylene is responsible for absence of internodal elongation in floating rice at low humidity.

Internodal elongation in floating rice (Oryza sativa) is known to be enhanced by treatment with ethylene or gibberellic acid (GA3) at high relative humidity (RH). However, ethylene-induced internodal elongation is inhibited at low RH, while GA3-induced internodal elongation is hardly affected by humidity. We examined the effects of ethylene and GA3 on the rate of transpiration in stem segments incubated at 30% or 100% RH. Ethylene promoted the transpiration of stem segments at 30% RH, but not at 100% RH, while GA3 had little effect on transpiration at either 30% or 100% RH. We propose that the absence of ethylene-induced internodal elongation at low RH is due, at least in part, to ethylene-induced transpiration.